Repair and regeneration of ocular tissue using postpartum-derived cells

ABSTRACT

Cells derived from postpartum umbilicus and placenta are disclosed. Pharmaceutical compositions, devices and methods for the regeneration or repair of ocular tissue using the postpartum-derived cells are also disclosed.

This a continuation in part of U.S. application Ser. No. 10/877,541,filed Jun. 25, 2004, the entire contents of which are incorporated byreference herein.

FIELD OF THE INVENTION

This invention relates to the field of cell-based or regenerativetherapy for ophthalmic diseases and disorders. In particular, theinvention provides pharmaceutical compositions, devices and methods forthe regeneration or repair of cells and tissues of the eye, usingpostpartum derived cells.

BACKGROUND

Various patents and other publications are referred to throughout thespecification. Each of these publications is incorporated by referenceherein, in its entirety.

As a complex and sensitive organ of the body, the eye can experiencenumerous diseases and other deleterious conditions that affect itsability to function normally. Many of these conditions are associatedwith damage or degeneration of specific ocular cells, and tissues madeup of those cells. As one example, diseases and degenerative conditionsof the optic nerve and retina are the leading causes of blindnessthroughout the world. Damage or degeneration of the cornea, lens andassociated ocular tissues represent another significant cause of visionloss worldwide.

The retina contains seven layers of alternating cells and processes thatconvert a light signal into a neural signal. The retinal photoreceptorsand adjacent retinal pigment epithelium (RPE) form a functional unitthat, in many disorders, becomes unbalanced due to genetic mutations orenvironmental conditions (including age). This results in loss ofphotoreceptors through apoptosis or secondary degeneration, which leadsto progressive deterioration of vision and, in some instances, toblindness (for a review, see, e.g., Lund, R. D. et al. 2001, Progress inRetinal and Eye Research 20: 415-449). Two classes of ocular disordersthat fall into this pattern are age-related macular degeneration (AMD)and retinitis pigmentosa (RP).

AMD is the most common cause of vision loss in the United States inthose people whose ages are 50 or older, and its prevalence increaseswith age. The primary disorder in AMD appears to be due to RPEdysfunction and changes in Bruch's membranes, characterized by, amongother things, lipid deposition, protein cross-linking and decreasedpermeability to nutrients (see Lund et al., 2001 supra). A variety ofelements may contribute to macular degeneration, including geneticmakeup, age, nutrition, smoking and exposure to sunlight.

AMD is broadly divided into two types. In the exudative-neovascularform, or “wet” AMD, which accounts for 10% of all cases, abnormal bloodvessel growth occurs under the macula. These blood vessels leak fluidand blood into the retina and thus cause damage to the photoreceptors.The remaining 90% of AMD cases are the nonexudative, or “dry” form. Inthese patients there is a gradual disappearance of the retinal pigmentepithelium (RPE), resulting in circumscribed areas of atrophy. Sincephotoreceptor loss follows the disappearance of RPE, the affectedretinal areas have little or no visual function.

For example, Radeke, M. et al state “Macular degeneration is a blindingdisease caused by the death of the photoreceptor cells in that part ofthe retina known as the macula (Radeke M et al, 2007). Radeke, M. et alfurther state, “Photoreceptors are critically dependent upon the RPEcells for their own survival” (Radeke M et al, 2007).

Current therapies for AMD involve procedures, such as, for example,laser therapy and pharmacological intervention. By transferring thermalenergy, the laser beam destroys the leaky blood vessels under themacula, which slows the rate of vision loss. A disadvantage of thisapproach, however, is that the high thermal energy delivered by the beamalso destroys healthy tissue nearby. Neuroscience 4^(th) edition,(Purves, D, et al 2008) states “Currently there is no treatment for dryAMD.”

RPE transplantation, following excision of choroid neovascularization isalso one potential cell-based therapy for AMD. However, this has beenunsuccessful in humans. For example, Zarbin, M, 2003 states, “Withnormal aging, human Bruch's membrane, especially in the submacularregion, undergoes numerous changes (e.g., increased thickness,deposition of ECM and lipids, cross-linking of protein, non-enzymaticformation of advanced glycation end products). These changes andadditional changes due to AMD could decrease the bioavailability of ECMligands (e.g., laminin, fibronectin, and collagen IV) and cause theextremely poor survival of RPE cells in eyes with AMD. Thus, althoughhuman RPE cells express the integrins needed to attach to these ECMmolecules, RPE cell survival on aged submacular human Bruch's membraneis impaired”.

RP is mainly considered an inherited disease, with over 100 mutationsbeing associated with photoreceptor loss (see Lund et al., 2001, supra).Though the majority of mutations target photoreceptors, some affect RPEcells directly. Together, these mutations affect such processes asmolecular trafficking between photoreceptors and RPE cells andphototransduction, for example.

Other less common, but nonetheless debilitating retinopathies can alsoinvolve progressive cellular degeneration leading to vision loss andblindness. These include, for example, diabetic retinopathy andchoroidal neovascular membrane (CNVM).

Diabetic retinopathy may be classified as (1) non-proliferative orbackground retinopathy, characterized by increased capillarypermeability, edema, hemorrhage, microaneurysms, and exudates, or 2)proliferative retinopathy, characterized by neovascularization extendingfrom the retina to the vitreous, scarring, fibrous tissue formation, andpotential for retinal detachment.

In CNVM, abnormal blood vessels stemming from the choroid grow upthrough the retinal layers. The fragile new vessels break easily,causing blood and fluid to pool within the layers of the retina.

Damage or progressive degeneration of the optic nerve and related nervesof the eye constitutes another leading cause of vision loss andblindness. A prime example is glaucoma, a condition of the eye that ismade up of a collection of eye diseases that cause vision loss by damageto the optic nerve. Elevated intraocular pressure (TOP) due toinadequate ocular drainage is a primary cause of glaucoma, but it canalso develop in the absence of elevated IOP. Glaucoma can develop as theeye ages. It can also occur as the result of an eye injury,inflammation, tumor, or in advanced cases of cataract or diabetes, or itcan be caused by certain drugs, such as, for example, steroids. Theprimary features of the optic neuropathy in glaucoma includecharacteristic changes in the optic nerve head, a decrease in number ofsurviving retinal ganglion cells, and loss of vision. It has beenproposed that a cascade of events links degeneration of the optic nervehead with the slow death of retinal ganglion cells observed in thedisease, and that this cascade of events can be slowed or preventedthrough the use of neuroprotective agents (Osborne et al., 2003, Eur. J.Ophthalmol. 13 (Supp 3): S19-S26).

Cellular damage and degenerative conditions also affect other parts ofthe eye. For example, cataracts result from gradual opacification of thecrystalline lens of the eye. It is believed that once begun, cataractdevelopment proceeds along one or more common pathways that culminate indamage to lens fibers. This condition is presently treated by surgicalremoval and replacement of the affected lens. Another example concernsthe cornea and surrounding conjuctiva that make up the ocular surface.The limbal epithelium, located between the cornea and the bulbarconjuctiva, contains corneal epithelial stem cells. Limbal epithelialcell deficiency (LECD) is a condition that occurs, for example, inStevens-Johnson syndrome and thermal or chemical burns. LECD often leadsto an imbalance between the corneal epithelium and the conjunctivalepithelium in which the cornea is covered by invading conjunctivalepithelial cells, which severely compromises the corneal surface andaffects visual acuity (Nakamura, T. & Kinoshita, S., 2003. Cornea 22(Supp. 1): S75-S80).

The recent advent of stem cell-based therapy for tissue repair andregeneration provides promising treatments for a number ofaforementioned cell-degenerative pathologies and other ocular disorders.Stem cells are capable of self-renewal and differentiation to generate avariety of mature cell lineages. Transplantation of such cells can beutilized as a clinical tool for reconstituting a target tissue, therebyrestoring physiologic and anatomic functionality. The application ofstem cell technology is wide-ranging, including tissue engineering, genetherapy delivery, and cell therapeutics, i.e., delivery ofbiotherapeutic agents to a target location via exogenously suppliedliving cells or cellular components that produce or contain those agents(For a review, see, for example, Tresco, P. A. et al., 2000, AdvancedDrug Delivery Reviews 42: 2-37).

An obstacle to realization of the therapeutic potential of stem celltechnology has been the difficulty in obtaining sufficient numbers ofstem cells. One source of stem cells is embryonic or fetal tissue.Embryonic stem and progenitor cells have been isolated from a number ofmammalian species, including humans, and several such cell types havebeen shown capable of self-renewal and expansion, as welldifferentiation into a variety of cell lineages. In animal modelsystems, embryonic stem cells have been reported to differentiate into aRPE cell phenotype, as well as to enhance the survival of hostphotoreceptors following transplantation (Haruta, M. et al., 2004,Investig. Ophthalmol. Visual Sci. 45: 1020-1025; Schraermeyer, U. etal., 2001, Cell Transplantation 10: 673-680). But the derivation of stemcells from embryonic or fetal sources has raised many ethical issuesthat are desirable to avoid by identifying other sources of multipotentor pluripotent cells.

Adult tissue also can yield stem cells useful for cell-based oculartherapy. For instance, retinal and corneal stem cells themselves may beutilized for cell replacement therapy in the eye. In addition, neuralstem cells from the hippocampus have been reported to integrate with thehost retina, adopting certain neural and glial characteristics (seereview of Lund, R. L. et al., 2003, J. Leukocyte Biol. 74: 151-160).Neural stem cells prepared from fetal rat cortex were shown todifferentiate along an RPE cell pathway following transplantation intothe adult rat subretinal space (Enzmann, V. et al., 2003, Investig.Ophthalmol. Visual Sci. 44: 5417-5422). Bone marrow stem cells have beenreported to differentiate into retinal neural cells and photoreceptorsfollowing transplantation into host retinas (Tomita, M. et al., 2002,Stem Cells 20: 279-283; Kicic, A. et al., 2003, J. Neurosci. 23:7742-7749). An ocular surface reconstruction in a rabbit model system,utilizing cultured mucosal epithelial stem cells, has also been reported(Nakamura, T. & Kinoshita, S., 2003, supra). While these reports showpromise for the use of adult progenitor and stem cells in cell-basedtherapy for the eye, it must be noted that adult stem cell populationsare comparatively rare and are often obtainable only by invasiveprocedures. Further, adult stem cells may have a more limited ability toexpand in culture than do embryonic stem cells.

Thus, a need exists for alternative sources of adequate supplies ofcells having the ability to support, augment or replace lost cellularfunction in the eye. A reliable, well-characterized and plentiful supplyof substantially homogeneous populations of such cells would be anadvantage in a variety of diagnostic and therapeutic applications inocular repair and regeneration, including drug screening assays, ex vivoor in vitro trophic support of ocular and other useful cell types, andin vivo cell-based therapy.

SUMMARY

This invention provides compositions and methods applicable tocell-based or regenerative therapy for ophthalmic diseases anddisorders. In particular, the invention features pharmaceuticalcompositions, devices and methods for the regeneration or repair ofocular tissue using postpartum-derived cells.

One aspect of the invention features an isolated postpartum-derivedcell, derived from human placental or umbilical cord tissuesubstantially free of blood, wherein the cell is capable of self-renewaland expansion in culture and has the potential to differentiate into acell of a neural phenotype; wherein the cell requires L-valine forgrowth and is capable of growth in at least about 5% oxygen. This cellfurther comprises one or more of the following characteristics: (a)potential for at least about 40 doublings in culture; (b) attachment andexpansion on a coated or uncoated tissue culture vessel, wherein thecoated tissue culture vessel comprises a coating of gelatin, laminin,collagen, polyomithine, vitronectin, or fibronectin; (c) production ofat least one of tissue factor, vimentin, and alpha-smooth muscle actin;(d) production of at least one of CD10, CD13, CD44, CD73, CD90,PDGFr-alpha, PD-L2 and HLA-A, B, C; (e) lack of production of at leastone of CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G,and HLA-DR, DP, DQ, as detected by flow cytometry; (f) expression of agene, which relative to a human cell that is a fibroblast, a mesenchymalstem cell, or an ileac crest bone marrow cell, is increased for at leastone of a gene encoding: interleukin 8; reticulon 1; chemokine (C--X--Cmotif) ligand 1 (melonoma growth stimulating activity, alpha); chemokine(C--X--C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine(C--X--C motif) ligand 3; tumor necrosis factor, alpha-induced protein3; C-type lectin superfamily member 2; Wilms tumor 1; aldehydedehydrogenase 1 family member A2; renin; oxidized low densitylipoprotein receptor 1; Homo sapiens clone IMAGE:4179671; protein kinaseC zeta; hypothetical protein DKFZp564F013; downregulated in ovariancancer 1; and Homo sapiens gene from clone DKFZp547k1113; (g) expressionof a gene, which relative to a human cell that is a fibroblast, amesenchymal stem cell, or an ileac crest bone marrow cell, is reducedfor at least one of a gene encoding: short stature homeobox 2; heatshock 27 kDa protein 2; chemokine (C--X--C motif) ligand 12 (stromalcell-derived factor 1); elastin (supravalvular aortic stenosis,Williams-Beuren syndrome); Homo sapiens mRNA; cDNA DKFZp586M2022 (fromclone DKFZp586M2022); mesenchyme homeo box 2 (growth arrest-specifichomeo box); sine oculis homeobox homolog 1 (Drosophila); crystallin,alpha B; disheveled associated activator of morphogenesis 2;DKFZP586B2420 protein; similar to neuralin 1; tetranectin (plasminogenbinding protein); src homology three (SH3) and cysteine rich domain;cholesterol 25-hydroxylase; runt-related transcription factor 3;interleukin 11 receptor, alpha; procollagen C-endopeptidase enhancer;frizzled homolog 7 (Drosophila); hypothetical gene BC008967; collagen,type VIII, alpha 1; tenascin C (hexabrachion); iroquois homeobox protein5; hephaestin; integrin, beta 8; synaptic vesicle glycoprotein 2;neuroblastoma, suppression of tumorigenicity 1; insulin-like growthfactor binding protein 2, 36 kDa; Homo sapiens cDNA FLJ12280 fis, cloneMAMMA1001744; cytokine receptor-like factor 1; potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 4; integrin, beta 7; transcriptional co-activator withPDZ-binding motif (TAZ); sine oculis homeobox homolog 2 (Drosophila);KIAA1034 protein; vesicle-associated membrane protein 5 (myobrevin);EGF-containing fibulin-like extracellular matrix protein 1; early growthresponse 3; distal-less homeo box 5; hypothetical protein FLJ20373;aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroiddehydrogenase, type II); biglycan; transcriptional co-activator withPDZ-binding motif (TAZ); fibronectin 1; proenkephalin; integrin,beta-like 1 (with EGF-like repeat domains); Homo sapiens mRNA fulllength insert cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein;natriuretic peptide receptor C/guanylate cyclase C (atrionatriureticpeptide receptor C); hypothetical protein FLJ14054; Homo sapiens mRNA;cDNA DKFZp564B222 (from clone DKFZp564B222); BCL2/adenovirus E1B 19 kDainteracting protein 3-like; AE binding protein 1; cytochrome c oxidasesubunit VIIa polypeptide 1 (muscle); similar to neuralin 1; B celltranslocation gene 1; hypothetical protein FLJ23191; and DKFZp586L151;(h) secretion of at least one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF,FGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1; and (i) lack ofsecretion of at least one of TGF-beta2, ANG2, PDGFbb, MIP1b, 1309, MDC,and VEGF, as detected by ELISA.

In certain embodiments, the postpartum-derived cell is anumbilicus-derived cell. In other embodiments it is a placenta-derivedcell. In specific embodiments, the cell has all identifying features ofany one of: cell type PLA 071003 (P8) (ATCC Accession No. PTA-6074);cell type PLA 071003 (P11) (ATCC Accession No. PTA-6075); cell type PLA071003 (P16) (ATCC Accession No. PTA-6079); cell type UMB 022803 (P7)(ATCC Accession No. PTA-6067); or cell type UMB 022803 (P17) (ATCCAccession No. PTA-6068).

In certain embodiments, postpartum-derived cells are isolated in thepresence of one or more enzyme activities comprising metalloproteaseactivity, mucolytic activity and neutral protease activity. Preferably,the cells have a normal karyotype, which is maintained as the cells arepassaged in culture. In preferred embodiments, the postpartum-derivedcells comprise each of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, andHLA-A, B, C and does not comprise any of CD31, CD34, CD45, CD117, CD141,or HLA-DR, DP, DQ, as detected by flow cytometry.

Another aspect of the invention features a cell population comprisingthe postpartum-derived cells as described above. In one embodiment, thepopulation is a substantially homogeneous population of thepostpartum-derived cells. In a specific embodiment, the populationcomprises a clonal cell line of the postpartum-derived cells. In anotherembodiment, the population is a heterogeneous population comprising thepostpartum-derived cells and at least one other cell type. In certainembodiments, the other cell type is an astrocyte, oligodendrocyte,neuron, neural progenitor, neural stem cell, retinal epithelial stemcell, corneal epithelial stem cell or other multipotent or pluripotentstem cell. In other embodiments, the cell population is cultured incontact with one or more factors that stimulate stem celldifferentiation toward a neural or epithelial lineage.

Also featured in accordance with the present invention is a cell lysateprepared from postpartum-derived cells. The cell lysate may be separatedinto a membrane enriched fraction and a soluble cell fraction. Theinvention also features an extracellular matrix produced by thepostpartum-derived cells, as well as a conditioned medium in which thecells have been grown.

Another aspect of the invention features a method of treating a patienthaving an ocular degenerative condition, which comprises administeringto the patient multipotent or pluripotent cells isolated from apostpartum placenta or umbilical cord, in an amount effective to treatthe ocular degenerative condition. Preferably the cells arepostpartum-derived cells, as described above. In certain embodiments,the ocular degenerative condition is an acute ocular degenerativecondition such as brain trauma, optic nerve trauma or ocular lesion. Inother embodiments, it is a chronic or progressive degenerativecondition, such as macular degeneration, retinitis pigmentosa, diabeticretinopathy, glaucoma or limbal epithelial cell deficiency. In certainembodiments, the cells are induced in vitro to differentiate into aneural or epithelial lineage cells prior to administration.

In certain embodiments, the cells are administered with at least oneother cell type, such as an astrocyte, oligodendrocyte, neuron, neuralprogenitor, neural stem cell, retinal epithelial stem cell, cornealepithelial stem cell, or other multipotent or pluripotent stem cell. Inthese embodiments, the other cell type can be administeredsimultaneously with, or before, or after, the postpartum-derived cells.Likewise, in these and other embodiments, the cells are administeredwith at least one other agent, such as a drug for ocular therapy, oranother beneficial adjunctive agent such as an anti-inflammatory agent,anti-apoptotic agents, antioxidants or growth factors. In theseembodiments, the other agent can be administered simultaneously with, orbefore, or after, the postpartum cells.

In various embodiments, the cells are administered to the surface of aneye, or they are administered to the interior of an eye or to a locationin proximity to the eye, e.g., behind the eye. The cells can beadministered through a cannula or from a device implanted in thepatient's body within or in proximity to the eye, or they may beadministered by implantation of a matrix or scaffold containing thecells.

Another aspect of the invention features a pharmaceutical compositionfor treating a patient having an ocular degenerative condition,comprising a pharmaceutically acceptable carrier and multipotent orpluripotent cells isolated from a postpartum placenta or umbilical cordin an amount effective to treat the ocular degenerative condition.Preferably the postpartum cells are postpartum-derived cells asdescribed above. The ocular degenerative condition may be an acute,chronic or progressive condition. In certain embodiments, thecomposition comprises cells that have been induced in vitro todifferentiate into a neural or epithelial lineage cells prior toformulation of the composition.

In certain embodiments, the pharmaceutical composition comprises atleast one other cell type, such as an astrocyte, oligodendrocyte,neuron, neural progenitor, neural stem cell, retinal epithelial stemcell, corneal epithelial stem cell, or other multipotent or pluripotentstem cell. In these or other embodiments, the pharmaceutical compositioncomprises at least one other agent, such as a drug for treating theocular degenerative disorder or other beneficial adjunctive agents,e.g., anti-inflammatory agents, anti-apoptotic agents, antioxidants orgrowth factors.

In certain embodiments, the pharmaceutical compositions are formulatedfor administration to the surface of an eye. Alternatively, they can beformulated for administration to the interior of an eye or in proximityto the eye (e.g., behind the eye). The compositions also can beformulated as a matrix or scaffold containing the cells.

According to yet another aspect of the invention, a kit is provided fortreating a patient having an ocular degenerative condition. The kitcomprises a pharmaceutically acceptable carrier, a population ofmultipotent or pluripotent cells isolated from postpartum placenta orumbilicus, preferably the postpartum-derived cells described above, andinstructions for using the kit in a method of treating the patient. Thekit may also contain one or more additional components, such as reagentsand instructions for culturing the cells, or a population of at leastone other cell type, or one or more agents useful in the treatment of anocular degenerative condition.

According to another aspect of the invention, a method is provided fortreating a patient having an ocular degenerative condition, whichcomprises administering to the patient a preparation made frommultipotent or pluripotent cells isolated from a postpartum placenta orumbilical cord, in an amount effective to treat the ocular degenerativecondition, wherein the preparation comprises a cell lysate of the cells(or fraction thereof), or a conditioned medium in which the cells weregrown, or an extracellular matrix of the cells. Preferably, the cellsare the postpartum-derived cells described above. In another aspect, theinvention features a pharmaceutical composition comprising apharmaceutically acceptable carrier and a preparation made from thepostpartum cells, which may be a cell lysate (or fraction thereof) ofthe postpartum cells, an extracellular matrix of the postpartum cells ora conditioned medium in which the postpartum cells were grown. Kits forpracticing this aspect of the invention are also provided. These mayinclude the one or more of a pharmaceutically acceptable carrier orother agent or reagent, one or more of a cell lysate or fractionthereof, an extracellular matrix or a conditioned medium from thepostpartum cells, and instructions for use of the kit components.

Another aspect of the invention features a method for increasing thesurvival, growth or activity of cells for transplantation to treat anocular degenerative disorder. The method comprises co-culturing thecells for transplantation with cultured cells derived from postpartumplacental or umbilical tissue, under conditions effective to increasethe survival, growth or activity of the cells for transplantation.Preferably, the postpartum cells are the postpartum-derived cellsdescribed above. A kit for practicing the method is also provided. Thekit comprises the cultured postpartum cells and instructions forco-culturing the cells for transplantation with the postpartum cellsunder conditions effective to increase the survival, growth or activityof the cells for transplantation.

In one embodiment, the present invention provides a method for treatinga patient having a retinopathy or a retinal/macular disorder, the methodcomprising administering to the patient's eye umbilicus-derived cells,in an amount effective to treat the retinopathy or a retinal/maculardisorder, wherein the cells are capable of self-renewal and expansion inculture, have the potential to differentiate into cells of at least aneural phenotype, and have the following characteristics:

-   -   a. Potential for at least 40 population doublings in culture;    -   b. Attachment and expansion on a coated or uncoated tissue        culture vessel, wherein the coated tissue culture vessel        comprises a coating of gelatin, laminin, collagen,        polyornithine, vitronectin, or fibronectin;    -   c. Production of vimentin and alpha-smooth muscle actin;    -   d. Production of CD10, CD13, CD44, CD73, and CD90; and    -   e. Expression of a gene, which is relative to a human cell that        is a fibroblast, a mesenchymal stem cell, or an ileac crest bone        marrow cell, is increased for a gene encoding interleukin 8 and        reticulon 1.

In one embodiment, the umbilicus-derived cells are positive for HLA-A,B, C, and negative for CD31, CD34, CD45, CD117, and CD141. In oneembodiment, the umbilicus-derived cells are expanded in culture prior toadministering to the patient's eye.

In one embodiment, the retinopathy or a retinal/macular disorder isage-related macular degeneration. In an alternate embodiment, theretinopathy or a retinal/macular disorder is glaucoma.

In one embodiment, the present invention provides a method forpreventing the loss of photoreceptor cells associated with a retinopathyor a retinal/macular disorder in a patient, the method comprisingadministering to the patient's eye umbilicus-derived cells, in an amounteffective to prevent the loss of photoreceptor cells, wherein the cellsare capable of self-renewal and expansion in culture, have the potentialto differentiate into cells of at least a neural phenotype, and have thefollowing characteristics:

-   -   a. Potential for at least 40 population doublings in culture;    -   b. Attachment and expansion on a coated or uncoated tissue        culture vessel, wherein the coated tissue culture vessel        comprises a coating of gelatin, laminin, collagen,        polyornithine, vitronectin, or fibronectin;    -   c. Production of vimentin and alpha-smooth muscle actin;    -   d. Production of CD10, CD13, CD44, CD73, and CD90; and    -   e. Expression of a gene, which is relative to a human cell that        is a fibroblast, a mesenchymal stem cell, or an ileac crest bone        marrow cell, is increased for a gene encoding interleukin 8 and        reticulon 1.

In one embodiment, the umbilicus-derived cells are positive for HLA-A,B, C, and negative for CD31, CD34, CD45, CD117, and CD141. In oneembodiment, the umbilicus-derived cells are expanded in culture prior toadministering to the patient's eye.

In one embodiment, the retinopathy or a retinal/macular disorder isage-related macular degeneration. In an alternate embodiment, theretinopathy or a retinal/macular disorder is glaucoma.

In one embodiment, the loss of photoreceptor cells is prevented byinhibiting the apoptosis of the photoreceptor cells. In an alternateembodiment, the loss of photoreceptor cells is prevented by stimulatingthe phagocytosis of shed photoreceptor fragments.

In one embodiment, the umbilicus-derived cells phagocytose the shedphotoreceptor fragments. In an alternate embodiment, theumbilicus-derived cells stimulate the phagocytosis of shed photoreceptorfragments by RPE cells.

In one embodiment, the loss of photoreceptor cells is prevented byreplacing the photoreceptor cells. In one embodiment, the photoreceptorcells are replaced by the umbilicus-derived cells differentiatingretinal progenitor cells into photoreceptor cells.

In one embodiment, the loss of photoreceptor cells is prevented byresurfacing Bruch's membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graphical representation of the layout of the cells in atypical trans-well experiment.

FIG. 2 shows the effect of umbilicus-derived cells on phagocytosisobserved in aged primary human RPE cells.

FIG. 3 shows the analysis of human dermal fibroblast stimulatedphagocytosis.

FIG. 4 shows the effects of umbilicus-derived cells on the phagocytosisof fluorescein isothiocyanate—photoreceptor outer segments observed innormal and dystrophic RPE cells.

FIG. 5 shows the adherence and integration of umbilicus-derived cells onaged explants of human Bruch's membrane after 7 days.

FIG. 6 shows the phototransduction pathway in cone and rod cells.Shading denotes up-regulated expression.

FIG. 7 shows the dose-dependant effect of H₂O₂-induced DNA fragmentationin ARPE-19 cells. Filled square and solid line: normal growth media;open square and dotted line: media conditioned using umbilicus-derivedcells.

FIG. 8 shows the dose-dependant effect of H₂O₂-induced early apoptosisin ARPE-19 cells. Upper panel: early apoptotic cells: Annexin V(+) and7-ADD (−); lower panel: total apoptotic cells: Annexin V(+). Filledsquare and solid line: normal growth media; open square and dotted line:conditioned media.

FIG. 9 shows the time course of H₂O₂-induced DNA fragmentation inARPE-19 cells. Filled square and solid line: normal growth media; opensquare and dotted line: media conditioned using umbilicus-derived cells.Dotted line: media conditioned using c umbilicus-derived cells.

FIG. 10 shows a representative image of an injected eye. This eye wasembedded in paraffin and sectioned until the suture was visible on astereomicroscope and the image was collected. The eye shown was avehicle-injected eye 1 day post-injection. The suture placed followingthe injection procedure can be seen on the right side of the eye(arrow).

FIG. 11 shows the morphometric analysis of the outer nuclear layer(ONL). Shown is an eye 60 days post-injection, 2× magnification. Theregions of ONL analysis are outlined in boxes. Region 1 (bottom box) islocal to the injection site, and region 2 (top box) is distal to theinjection site, on the opposite side of the eye.

FIG. 12 shows the identification of umbilicus-derived cells insubretinal space, day 1 post-injection. Panel A: H&E stained section ofa day 1 eye at 20× magnification. Injected umbilicus-derived cells canbe seen in subretinal space. Panel B: an adjacent section immunostainedfor NuMA, 40× magnification. Infiltrating neutrophils can also beidentified amongst NuMA-positive umbilicus-derived cells.

FIG. 13 shows cell retention in the RCS rat eye. Cell retention in theRCS rat eyes from Day 1 to Day 60; the Y-axis is on logarithmic scale.Human P2M mRNA was detected in eyes 1, 7, 14, and 60 days aftersubretinal injection. The cell number reduced gradually from 18227±3227(mean+/−standard error of the mean; n=4) at Day 1 to 2266±1328(mean+/−standard error of the mean; n=4) at Day 60.

FIG. 14 shows the time course of ONL degeneration. H&E stained sectionsof control and umbilicus-derived cell injected eyes from the followingtime points post-injection: Day 7 (A and B), Day 14 (C and D), Day 30 (Eand F), and Day 60 (G and H). Images were acquired using 40×magnification near the injection site region. Arrows mark the outernuclear layer.

FIG. 15 demonstrates the injection of umbilicus-derived cells preservesONL thickness at Day 60 post-injection. Representative images from H&Estained sections from umbilicus-derived cell injected and control eyes60 days post-injection, 60× magnification. Panels A and B: Control eye,images taken from areas near and far from injection site, respectively.ONL (arrows) appears as a single discontinuous layer in both regions.Panels C and D: umbilicus-derived cell injected eye, areas near and farfrom injection site, respectively. ONL is visibly thicker in bothregions, although the area near the injection site is thicker than thatfurther away (approximately 4 nuclei thick near injection site comparedto 2 nuclei thick far from injection site in these images). Panels E andF: morphometric results. Panel E: All data from both regions analyzed(near to and far from injection site) were combined for each animal (n=3animals per group). Values graphed are the mean+/−standard deviation.Means were significantly different between control eyes and eyesinjected with umbilicus-derived cells (p<0.001, t-test). Panel F:Regional ONL rescue. The two columns on the left side of the graph arefrom region 1 (close to injection site), the two columns on the rightside are from region 2 (far from the injection site). Values graphed arethe mean+/−standard deviation of all tissue sections analyzed (forregion 1, the number of images were n=6 and 9 and region 2, the numberof images were n=9 and 6 for control and umbilicus-derived cells,respectively). The means for both groups were significantly different ineach region (p=0.002 using Mann-Whitney rank sum test for region 1, andp<0.001 using t-test for region 2. Mann-Whitney test was used for region1 because the umbilicus-derived cells group did not have a normaldistribution).

FIG. 16 demonstrates injection of umbilicus-derived cells preservesrhodopsin immunostaining at Day 60 post-injection. Panels A and B:representative images from Day 60 control and umbilicus-derived cellinjected animals at 60× magnification near the injection site. Panel A:control, panel B: umbilicus-derived cells. Rhodopsin immunostaining isspecific to the neuroepithelial layer (arrows) and is visibly increasedin the umbilicus-derived cell injected eye compared with control. PanelC: morphometric results, values graphed are the mean+/−standarddeviation (n=3 animals per group). Means were significantly differentbetween control eyes and eyes injected with umbilicus-derived cells:7.04+/−5.96 and 72.83+/−16.63, respectively (p=0.003, t-test). Valuesrepresent raw data multiplied by 100 to be expressed as a percentage.

FIG. 17 demonstrates injection of umbilicus-derived cells preservescalretinin immunostaining at Day 60 post-injection. Shown arerepresentative images from Day 60 control and umbilicus-derived cellinjected animals at 60× magnification near the injection site. Panel A:control, panel B: umbilicus-derived cells. Calretinin immunostaining isspecific to the inner nuclear layer (INL), inner plexiform layer (IPL),and the retinal ganglion cell layer (GCL). Panels C and D: morphometricresults, values graphed are the mean+/−standard deviation (n=3 animalsper group). Panel C: total calretinin staining (GCL, IPL, INL). Meanswere significantly different between control eyes and eyes injected withumbilicus-derived cells: 10.05+/−0.66 and 12.52+/−0.60, respectively(p=0.009, t-test). Panel D: calretinin staining of INL and GCL. Meanswere significantly different between control and umbilicus-derived cellinjected eyes in the INL but not in the GCL: 3.82+/−0.22 and 5.67+/−0.85(INL, p=0.014, t-test) and 2.91+/−1.11 and 3.16+/−0.37 (GCL, p=0.629,t-test), respectively.

FIG. 18 demonstrates injection of umbilicus-derived cells preservesrecoverin immunostaining at Day 60 post-injection. umbilicus-derivedcells significantly preserves recoverin immunostaining at day 60post-injection. Panels A and B: representative images from Day 60control and umbilicus-derived cell injected animals at 60× magnificationnear the injection site. Panel A: control, panel B: umbilicus-derivedcells. Recoverin immunostaining is specific to the outer nuclear layer(arrows) and a subpopulation of cells in the inner nuclear layer (arrowheads). Panels C and D: morphometric results, values graphed are themean+/−standard deviation (n=3 animals per group). Panel C: totalrecoverin staining (INL, ONL). Means were significantly differentbetween control eyes and eyes injected with umbilicus-derived cells:2.47+/−0.50 and 13.03+/−3.28, respectively (P=0.001, t-test). Panel D:recoverin staining of INL and ONL. Means were significantly differentbetween control and umbilicus-derived cell injected eyes in both the INLand ONL 1.11+/−0.31 and 2.24+/−0.51 (INL, p=0.031, t-test) and1.36+/−0.34 and 10.78+/−2.87 (ONL, p=0.001, t-test), respectively.

Other features and advantages of the invention will be apparent from thedetailed description and examples that follow.

DETAILED DESCRIPTION

Various patents and other publications are referred to throughout thespecification. Each of these publications is incorporated by referenceherein, in its entirety.

DEFINITIONS

Various terms used throughout the specification and claims are definedas set forth below.

Stem cells are undifferentiated cells defined by the ability of a singlecell both to self-renew, and to differentiate to produce progeny cells,including self-renewing progenitors, non-renewing progenitors, andterminally differentiated cells. Stem cells are also characterized bytheir ability to differentiate in vitro into functional cells of variouscell lineages from multiple germ layers (endoderm, mesoderm andectoderm), as well as to give rise to tissues of multiple germ layersfollowing transplantation, and to contribute substantially to most, ifnot all, tissues following injection into blastocysts.

Stem cells are classified according to their developmental potential as:(1) totipotent; (2) pluripotent; (3) multipotent; (4) oligopotent; and(5) unipotent. Totipotent cells are able to give rise to all embryonicand extraembryonic cell types. Pluripotent cells are able to give riseto all embryonic cell types. Multipotent cells include those able togive rise to a subset of cell lineages, but all within a particulartissue, organ, or physiological system (for example, hematopoietic stemcells (HSC) can produce progeny that include HSC (self-renewal), bloodcell-restricted oligopotent progenitors, and all cell types and elements(e.g., platelets) that are normal components of the blood). Cells thatare oligopotent can give rise to a more restricted subset of celllineages than multipotent stem cells; and cells that are unipotent areable to give rise to a single cell lineage (e.g., spermatogenic stemcells).

Stem cells are also categorized on the basis of the source from whichthey may be obtained. An adult stem cell is generally a multipotentundifferentiated cell found in tissue comprising multiple differentiatedcell types. The adult stem cell can renew itself. Under normalcircumstances, it can also differentiate to yield the specialized celltypes of the tissue from which it originated, and possibly other tissuetypes. An embryonic stem cell is a pluripotent cell from the inner cellmass of a blastocyst-stage embryo. A fetal stem cell is one thatoriginates from fetal tissues or membranes. A postpartum stem cell is amultipotent or pluripotent cell that originates substantially fromextraembryonic tissue available after birth, namely, the placenta andthe umbilical cord. These cells have been found to possess featurescharacteristic of pluripotent stem cells, including rapid proliferationand the potential for differentiation into many cell lineages.Postpartum stem cells may be blood-derived (e.g., as are those obtainedfrom umbilical cord blood) or non-blood-derived (e.g., as obtained fromthe non-blood tissues of the umbilical cord and placenta).

Embryonic tissue is typically defined as tissue originating from theembryo (which in humans refers to the period from fertilization to aboutsix weeks of development. Fetal tissue refers to tissue originating fromthe fetus, which in humans refers to the period from about six weeks ofdevelopment to parturition. Extraembryonic tissue is tissue associatedwith, but not originating from, the embryo or fetus. Extraembryonictissues include extraembryonic membranes (chorion, amnion, yolk sac andallantois), umbilical cord and placenta (which itself forms from thechorion and the maternal decidua basalis).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cell,such as a nerve cell or a muscle cell, for example. A differentiatedcell is one that has taken on a more specialized (“committed”) positionwithin the lineage of a cell. The term committed, when applied to theprocess of differentiation, refers to a cell that has proceeded in thedifferentiation pathway to a point where, under normal circumstances, itwill continue to differentiate into a specific cell type or subset ofcell types, and cannot, under normal circumstances, differentiate into adifferent cell type or revert to a less differentiated cell type.De-differentiation refers to the process by which a cell reverts to aless specialized (or committed) position within the lineage of a cell.As used herein, the lineage of a cell defines the heredity of the cell,i.e. which cells it came from and what cells it can give rise to. Thelineage of a cell places the cell within a hereditary scheme ofdevelopment and differentiation.

In a broad sense, a progenitor cell is a cell that has the capacity tocreate progeny that are more differentiated than itself, and yet retainsthe capacity to replenish the pool of progenitors. By that definition,stem cells themselves are also progenitor cells, as are the moreimmediate precursors to terminally differentiated cells. When referringto the cells of the present invention, as described in greater detailbelow, this broad definition of progenitor cell may be used. In anarrower sense, a progenitor cell is often defined as a cell that isintermediate in the differentiation pathway, i.e., it arises from a stemcell and is intermediate in the production of a mature cell type orsubset of cell types. This type of progenitor cell is generally not ableto self-renew. Accordingly, if this type of cell is referred to herein,it will be referred to as a non-renewing progenitor cell or as anintermediate progenitor or precursor cell.

As used herein, the phrase differentiates into an ocular lineage orphenotype refers to a cell that becomes partially or fully committed toa specific ocular phenotype, including without limitation, retinal andcorneal stem cells, pigment epithelial cells of the retina and iris,photoreceptors, retinal ganglia and other optic neural lineages (e.g.,retinal glia, microglia, astrocytes, Mueller cells), cells forming thecrystalline lens, and epithelial cells of the sclera, cornea, limbus andconjunctiva. The phrase differentiates into a neural lineage orphenotype refers to a cell that becomes partially or fully committed toa specific neural phenotype of the CNS or PNS, i.e., a neuron or a glialcell, the latter category including without limitation astrocytes,oligodendrocytes, Schwann cells and microglia.

The cells exemplified herein and preferred for use in present inventionare generally referred to as postpartum-derived cells (or PPDCs). Theyalso may sometimes be referred to more specifically as umbilicus-derivedcells or placenta-derived cells (UDCs or PDCs). In addition, the cellsmay be described as being stem or progenitor cells, the latter termbeing used in the broad sense. The term derived is used to indicate thatthe cells have been obtained from their biological source and grown invitro (e.g., cultured in a Growth Medium to expand the population and/orto produce a cell line). The in vitro manipulations of umbilical stemcells and the unique features of the umbilicus-derived cells of thepresent invention are described in detail below. Cells isolated frompostpartum placenta and umbilicus by other means is also consideredsuitable for use in the present invention. These other cells arereferred to herein as postpartum cells (rather than postpartum-derivedcells).

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition (“in culture” or “cultured”). A primary cellculture is a culture of cells, tissues, or organs taken directly from anorganism(s) before the first subculture. Cells are expanded in culturewhen they are placed in a Growth Medium under conditions that facilitatecell growth and/or division, resulting in a larger population of thecells. When cells are expanded in culture, the rate of cellproliferation is sometimes measured by the amount of time needed for thecells to double in number. This is referred to as doubling time.

A cell line is a population of cells formed by one or moresubcultivations of a primary cell culture. Each round of subculturing isreferred to as a passage. When cells are subcultured, they are referredto as having been passaged. A specific population of cells, or a cellline, is sometimes referred to or characterized by the number of timesit has been passaged. For example, a cultured cell population that hasbeen passaged ten times may be referred to as a P10 culture. The primaryculture, i.e., the first culture following the isolation of cells fromtissue, is designated P0. Following the first subculture, the cells aredescribed as a secondary culture (P1 or passage 1). After the secondsubculture, the cells become a tertiary culture (P2 or passage 2), andso on. It will be understood by those of skill in the art that there maybe many population doublings during the period of passaging; thereforethe number of population doublings of a culture is greater than thepassage number. The expansion of cells (i.e., the number of populationdoublings) during the period between passaging depends on many factors,including but not limited to the seeding density, substrate, medium,growth conditions, and time between passaging.

A conditioned medium is a medium in which a specific cell or populationof cells has been cultured, and then removed. When cells are cultured ina medium, they may secrete cellular factors that can provide trophicsupport to other cells. Such trophic factors include, but are notlimited to hormones, cytokines, extracellular matrix (ECM), proteins,vesicles, antibodies, and granules. The medium containing the cellularfactors is the conditioned medium.

Generally, a trophic factor is defined as a substance that promotessurvival, growth, differentiation, proliferation and/or maturation of acell, or stimulates increased activity of a cell. The interactionbetween cells via trophic factors may occur between cells of differenttypes. Cell interaction by way of trophic factors is found inessentially all cell types, and is a particularly significant means ofcommunication among neural cell types. Trophic factors also can functionin an autocrine fashion, i.e., a cell may produce trophic factors thataffect its own survival, growth, differentiation, proliferation and/ormaturation.

When referring to cultured vertebrate cells, the term senescence (alsoreplicative senescence or cellular senescence) refers to a propertyattributable to finite cell cultures; namely, their inability to growbeyond a finite number of population doublings (sometimes referred to asHayflick's limit). Although cellular senescence was first describedusing fibroblast-like cells, most normal human cell types that can begrown successfully in culture undergo cellular senescence. The in vitrolifespan of different cell types varies, but the maximum lifespan istypically fewer than 100 population doublings (this is the number ofdoublings for all the cells in the culture to become senescent and thusrender the culture unable to divide). Senescence does not depend onchronological time, but rather is measured by the number of celldivisions, or population doublings, the culture has undergone. Thus,cells made quiescent by removing essential growth factors are able toresume growth and division when the growth factors are re-introduced,and thereafter carry out the same number of doublings as equivalentcells grown continuously. Similarly, when cells are frozen in liquidnitrogen after various numbers of population doublings and then thawedand cultured, they undergo substantially the same number of doublings ascells maintained unfrozen in culture. Senescent cells are not dead ordying cells; they are actually resistant to programmed cell death(apoptosis), and have been maintained in their nondividing state for aslong as three years. These cells are alive and metabolically active, butthey do not divide. The nondividing state of senescent cells has not yetbeen found to be reversible by any biological, chemical, or viral agent.

The terms ocular, ophthalmic and optic are used interchangeably hereinto define “of, or about, or related to the eye.”

The term ocular degenerative condition (or disorder) is an inclusiveterm encompassing acute and chronic conditions, disorders or diseases ofthe eye, inclusive of the neural connection between the eye and thebrain, involving cell damage, degeneration or loss. An oculardegenerative condition may be age-related, or it may result from injuryor trauma, or it may be related to a specific disease or disorder. Acuteocular degenerative conditions include, but are not limited to,conditions associated with cell death or compromise affecting the eyeincluding conditions arising from cerebrovascular insufficiency, focalor diffuse brain trauma, diffuse brain damage, infection or inflammatoryconditions of the eye, retinal tearing or detachment, intra-ocularlesions (contusion penetration, compression, laceration) or otherphysical injury (e.g., physical or chemical burns). Chronic oculardegenerative conditions (including progressive conditions) include, butare not limited to, retinopathies and other retinal/macular disorderssuch as retinitis pigmentosa (RP), age-related macular degeneration(AMD), choroidal neovascular membrane (CNVM); retinopathies such asdiabetic retinopathy, occlusive retinopathy, sickle cell retinopathy andhypertensive retinopathy, central retinal vein occlusion, stenosis ofthe carotid artery, optic neuropathies such as glaucoma and relatedsyndromes; disorders of the lens and outer eye, e.g., limbal stem celldeficiency (LSCD), also referred to as limbal epithelial cell deficiency(LECD), such as occurs in chemical or thermal injury, Steven-Johnsonsyndrome, contact lens-induced keratopathy, ocular cicatricialpemphigoid, congenital diseases of aniridia or ectodermal dysplasia, andmultiple endocrine deficiency-associated keratitis.

The term treating (or treatment of) an ocular degenerative conditionrefers to ameliorating the effects of, or delaying, halting or reversingthe progress of, or delaying or preventing the onset of, an oculardegenerative condition as defined herein.

The term effective amount refers to a concentration or amount of areagent or pharmaceutical composition, such as a growth factor,differentiation agent, trophic factor, cell population or other agent,that is effective for producing an intended result, including cellgrowth and/or differentiation in vitro or in vivo, or treatment ofocular degenerative conditions, as described herein. With respect togrowth factors, an effective amount may range from about 1nanogram/milliliter to about 1 microgram/milliliter. With respect toPPDCs as administered to a patient in vivo, an effective amount mayrange from as few as several hundred or fewer, to as many as severalmillion or more. In specific embodiments, an effective amount may rangefrom 10³ to 10¹¹, more specifically at least about 10⁴ cells. It will beappreciated that the number of cells to be administered will varydepending on the specifics of the disorder to be treated, including butnot limited to size or total volume/surface area to be treated, as wellas proximity of the site of administration to the location of the regionto be treated, among other factors familiar to the medicinal biologist.

The terms effective period (or time) and effective conditions refer to aperiod of time or other controllable conditions (e.g., temperature,humidity for in vitro methods), necessary or preferred for an agent orpharmaceutical composition to achieve its intended result.

The term patient or subject refers to animals, including mammals,preferably humans, who are treated with the pharmaceutical compositionsor in accordance with the methods described herein.

The term pharmaceutically acceptable carrier (or medium), which may beused interchangeably with the term biologically compatible carrier ormedium, refers to reagents, cells, compounds, materials, compositions,and/or dosage forms that are not only compatible with the cells andother agents to be administered therapeutically, but also are, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other complication commensurate with areasonable benefit/risk ratio.

Several terms are used herein with respect to cell replacement therapy.The terms autologous transfer, autologous transplantation, autograft andthe like refer to treatments wherein the cell donor is also therecipient of the cell replacement therapy. The terms allogeneictransfer, allogeneic transplantation, allograft and the like refer totreatments wherein the cell donor is of the same species as therecipient of the cell replacement therapy, but is not the sameindividual. A cell transfer in which the donor's cells and have beenhistocompatibly matched with a recipient is sometimes referred to as asyngeneic transfer. The terms xenogeneic transfer, xenogeneictransplantation, xenograft and the like refer to treatments wherein thecell donor is of a different species than the recipient of the cellreplacement therapy. Transplantation as used herein refers to theintroduction of autologous, or allogeneic donor cell replacement therapyinto a recipient.

DESCRIPTION

Ocular degenerative conditions, which encompass acute, chronic andprogressive disorders and diseases having divergent causes, have as acommon feature the dysfunction or loss of a specific or vulnerable groupof ocular cells. This commonality enables development of similartherapeutic approaches for the repair or regeneration of vulnerable,damaged or lost ocular tissue, one of which is cell-based therapy.Development of cell therapy for ocular degenerative conditionsheretofore has been limited to a comparatively few types of stem orprogenitor cells, including ocular-derived stem cells themselves (e.g.,retinal and corneal stem cells), embryonic stem cells and a few types ofadult stem or progenitor cells (e.g., neural, mucosal epithelial andbone marrow stem cells). The present inventors have identified asignificant new source of stem cells for this purpose, namely, cellsisolated from the postpartum placenta and umbilical cord. Accordingly,in its various embodiments described herein, the present inventionfeatures methods and pharmaceutical compositions for repair andregeneration of ocular tissues, which utilize progenitor cells and cellpopulations isolated from postpartum tissues. The invention isapplicable to any ocular degenerative condition, but is expected to beparticularly suitable for a number of ocular disorders for whichtreatment or cure heretofore has been difficult or unavailable. Theseinclude, without limitation, age-related macular degeneration, retinitispigmentosa, diabetic and other retinopathies, glaucoma and other opticneuropathies, and disorders associated with limbal stem cell deficiency.

Stem or progenitor cells isolated from postpartum placenta or umbilicalcord in accordance with any method known in the art are expected to besuitable for use in the present invention. In a one embodiment, however,the invention utilizes postpartum-derived cells (PPDCs) as definedabove, which are derived from placental or umbilical cord tissue thathas been rendered substantially free of blood, preferably in accordancewith the method set forth below. The PPDCs are capable of self-renewaland expansion in culture and have the potential to differentiate intocells of other phenotypes. Certain embodiments features populationscomprising such cells, pharmaceutical compositions comprising the cellsor components or products thereof, and methods of using thepharmaceutical compositions for treatment of patients with acute orchronic ocular degenerative conditions. The postpartum-derived cells ofthe present invention have been characterized by their growth propertiesin culture, by their cell surface markers, by their gene expression, bytheir ability to produce certain biochemical trophic factors, and bytheir immunological properties.

Preparation of PPDCs

According to the methods described herein, a mammalian placenta andumbilical cord are recovered upon or shortly after termination of eithera full-term or pre-term pregnancy, for example, after expulsion afterbirth. The postpartum tissue may be transported from the birth site to alaboratory in a sterile container such as a flask, beaker, culture dish,or bag. The container may have a solution or medium, including but notlimited to a salt solution, such as, for example, Dulbecco's ModifiedEagle's Medium (DMEM) or phosphate buffered saline (PBS), or anysolution used for transportation of organs used for transplantation,such as University of Wisconsin solution or perfluorochemical solution.One or more antibiotic and/or antimycotic agents, such as but notlimited to penicillin, streptomycin, amphotericin B, gentamicin, andnystatin, may be added to the medium or buffer. The postpartum tissuemay be rinsed with an anticoagulant solution such as heparin-containingsolution. It is preferable to keep the tissue at about 4-10° C. prior toextraction of PPDCs. It is even more preferable that the tissue not befrozen prior to extraction of PPDCs.

Isolation of PPDCs preferably occurs in an aseptic environment. Theumbilical cord may be separated from the placenta by means known in theart. Alternatively, the umbilical cord and placenta are used withoutseparation. Blood and debris are preferably removed from the postpartumtissue prior to isolation of PPDCs. For example, the postpartum tissuemay be washed with buffer solution, such as but not limited to phosphatebuffered saline. The wash buffer also may comprise one or moreantimycotic and/or antibiotic agents, such as but not limited topenicillin, streptomycin, amphotericin B, gentamicin, and nystatin.

Postpartum tissue comprising a whole placenta or a fragment or sectionthereof is disaggregated by mechanical force (mincing or shear forces).In a presently preferred embodiment, the isolation procedure alsoutilizes an enzymatic digestion process. Many enzymes are known in theart to be useful for the isolation of individual cells from complextissue matrices to facilitate growth in culture. Ranging from weaklydigestive (e.g. deoxyribonucleases and the neutral protease, dispase) tostrongly digestive (e.g. papain and trypsin), such enzymes are availablecommercially. A nonexhaustive list of enzymes compatible herewithincludes mucolytic enzyme activities, metalloproteases, neutralproteases, serine proteases (such as trypsin, chymotrypsin, orelastase), and deoxyribonucleases. Presently preferred are enzymeactivities selected from metalloproteases, neutral proteases andmucolytic activities. For example, collagenases are known to be usefulfor isolating various cells from tissues. Deoxyribonucleases can digestsingle-stranded DNA and can minimize cell clumping during isolation.Preferred methods involve enzymatic treatment with for examplecollagenase and dispase, or collagenase, dispase, and hyaluronidase, andsuch methods are provided wherein in certain preferred embodiments, amixture of collagenase and the neutral protease dispase are used in thedissociating step. More preferred are those methods that employdigestion in the presence of at least one collagenase from Clostridiumhistolyticum, and either of the protease activities, dispase andthermolysin. Still more preferred are methods employing digestion withboth collagenase and dispase enzyme activities. Also preferred aremethods that include digestion with a hyaluronidase activity in additionto collagenase and dispase activities. The skilled artisan willappreciate that many such enzyme treatments are known in the art forisolating cells from various tissue sources. For example, the LIBERASEBlendzyme (Roche) series of enzyme combinations are suitable for use inthe instant methods. Other sources of enzymes are known, and the skilledartisan may also obtain such enzymes directly from their naturalsources. The skilled artisan is also well equipped to assess new, oradditional enzymes or enzyme combinations for their utility in isolatingthe cells of the invention. Preferred enzyme treatments are 0.5, 1, 1.5,or 2 hours long or longer. In other preferred embodiments, the tissue isincubated at 37° C. during the enzyme treatment of the dissociationstep.

In some embodiments of the invention, postpartum tissue is separatedinto sections comprising various aspects of the tissue, such asneonatal, neonatal/maternal, and maternal aspects of the placenta, forinstance. The separated sections then are dissociated by mechanicaland/or enzymatic dissociation according to the methods described herein.Cells of neonatal or maternal lineage may be identified by any meansknown in the art, for example, by karyotype analysis or in situhybridization for a Y chromosome.

Isolated cells or postpartum tissue from which PPDCs grow out may beused to initiate, or seed, cell cultures. Isolated cells are transferredto sterile tissue culture vessels either uncoated or coated withextracellular matrix or ligands such as laminin, collagen (native,denatured or crosslinked), gelatin, fibronectin, and other extracellularmatrix proteins. PPDCs are cultured in any culture medium capable ofsustaining growth of the cells such as, but not limited to, DMEM (highor low glucose), advanced DMEM, DMEM/MCDB 201, Eagle's basal medium,Ham's F10 medium (F10), Ham's F-12 medium (F12), Iscove's modifiedDulbecco's medium, Mesenchymal Stem Cell Growth Medium (MSCGM),DMEM/F12, RPMI 1640, and CELL-GRO-FREE. The culture medium may besupplemented with one or more components including, for example, fetalbovine serum (FBS), preferably about 2-15% (v/v); equine serum (ES);human serum (HS); beta-mercaptoethanol (BME or 2-ME), preferably about0.001% (v/v); one or more growth factors, for example, platelet-derivedgrowth factor (PDGF), epidermal growth factor (EGF), fibroblast growthfactor (FGF), vascular endothelial growth factor (VEGF), insulin-likegrowth factor-1 (IGF-1), leukocyte inhibitory factor (LIF) anderythropoietin; amino acids, including L-valine; and one or moreantibiotic and/or antimycotic agents to control microbial contamination,such as, for example, penicillin G, streptomycin sulfate, amphotericinB, gentamicin, and nystatin, either alone or in combination. The culturemedium preferably comprises Growth Medium (DMEM—low glucose, serum, BME,and an antibiotic agent).

The cells are seeded in culture vessels at a density to allow cellgrowth. In a preferred embodiment, the cells are cultured at about 0 toabout 5 percent by volume CO₂ in air. In some preferred embodiments, thecells are cultured at about 2 to about 25 percent O₂ in air, preferablyabout 5 to about 20 percent O₂ in air. The cells preferably are culturedat about 25 to about 40° C. and more preferably are cultured at 37° C.The cells are preferably cultured in an incubator. The medium in theculture vessel can be static or agitated, for example, using abioreactor. PPDCs preferably are grown under low oxidative stress (e.g.,with addition of glutathione, Vitamin C, Catalase, Vitamin E,N-Acetylcysteine). “Low oxidative stress”, as used herein, refers toconditions of no or minimal free radical damage to the cultured cells.

Methods for the selection of the most appropriate culture medium, mediumpreparation, and cell culture techniques are well known in the art andare described in a variety of sources, including Doyle et al., (eds.),1995, CELL & TISSUE CULTURE: LABORATORY PROCEDURES, John Wiley & Sons,Chichester; and Ho and Wang (eds.), 1991, ANIMAL CELL BIOREACTORS,Butterworth-Heinemann, Boston, which are incorporated herein byreference.

After culturing the isolated cells or tissue fragments for a sufficientperiod of time, PPDCs will have grown out, either as a result ofmigration from the postpartum tissue or cell division, or both. In someembodiments of the invention, PPDCs are passaged, or removed to aseparate culture vessel containing fresh medium of the same or adifferent type as that used initially, where the population of cells canbe mitotically expanded. The cells of the invention may be used at anypoint between passage 0 and senescence. The cells preferably arepassaged between about 3 and about 25 times, more preferably arepassaged about 4 to about 12 times, and preferably are passaged 10 or 11times. Cloning and/or subcloning may be performed to confirm that aclonal population of cells has been isolated.

In some aspects of the invention, the different cell types present inpostpartum tissue are fractionated into subpopulations from which thePPDCs can be isolated. This may be accomplished using standardtechniques for cell separation including, but not limited to, enzymatictreatment to dissociate postpartum tissue into its component cells,followed by cloning and selection of specific cell types, for examplebut not limited to selection based on morphological and/or biochemicalmarkers; selective growth of desired cells (positive selection),selective destruction of unwanted cells (negative selection); separationbased upon differential cell agglutinability in the mixed population as,for example, with soybean agglutinin; freeze-thaw procedures;differential adherence properties of the cells in the mixed population;filtration; conventional and zonal centrifugation; centrifugalelutriation (counter-streaming centrifugation); unit gravity separation;countercurrent distribution; electrophoresis; and fluorescence activatedcell sorting (FACS). For a review of clonal selection and cellseparation techniques, see Freshney, 1994, CULTURE OF ANIMAL CELLS: AMANUAL OF BASIC TECHNIQUES, 3rd Ed., Wiley-Liss, Inc., New York, whichis incorporated herein by reference.

The culture medium is changed as necessary, for example, by carefullyaspirating the medium from the dish, for example, with a pipette, andreplenishing with fresh medium. Incubation is continued until asufficient number or density of cells accumulates in the dish. Theoriginal explanted tissue sections may be removed and the remainingcells trypsinized using standard techniques or using a cell scraper.After trypsinization, the cells are collected, removed to fresh mediumand incubated as above. In some embodiments, the medium is changed atleast once at approximately 24 hours post-trypsinization to remove anyfloating cells. The cells remaining in culture are considered to bePPDCs.

PPDCs may be cryopreserved. Accordingly, in a preferred embodimentdescribed in greater detail below, PPDCs for autologous transfer (foreither the mother or child) may be derived from appropriate postpartumtissues following the birth of a child, then cryopreserved so as to beavailable in the event they are later needed for transplantation.

Characteristics of PPDCs

PPDCs may be characterized, for example, by growth characteristics(e.g., population doubling capability, doubling time, passages tosenescence), karyotype analysis (e.g., normal karyotype; maternal orneonatal lineage), flow cytometry (e.g., FACS analysis),immunohistochemistry and/or immunocytochemistry (e.g., for detection ofepitopes), gene expression profiling (e.g., gene chip arrays; polymerasechain reaction (for example, reverse transcriptase PCR, real time PCR,and conventional PCR)), protein arrays, protein secretion (e.g., byplasma clotting assay or analysis of PDC-conditioned medium, forexample, by Enzyme Linked ImmunoSorbent Assay (ELISA)), mixed lymphocytereaction (e.g., as measure of stimulation of PBMCs), and/or othermethods known in the art.

Examples of PPDCs derived from placental tissue were deposited with theAmerican Type Culture Collection (ATCC, Manassas, Va.) and assigned ATCCAccession Numbers as follows: (1) strain designation PLA 071003 (P8) wasdeposited Jun. 15, 2004 and assigned Accession No. PTA-6074; (2) straindesignation PLA 071003 (P11) was deposited Jun. 15, 2004 and assignedAccession No. PTA-6075; and (3) strain designation PLA 071003 (P16) wasdeposited Jun. 16, 2004 and assigned Accession No. PTA-6079. Examples ofPPDCs derived from umbilicus tissue were deposited with the AmericanType Culture Collection on Jun. 10, 2004, and assigned ATCC AccessionNumbers as follows: (1) strain designation UMB 022803 (P7) was assignedAccession No. PTA-6067; and (2) strain designation UMB 022803 (P17) wasassigned Accession No. PTA-6068.

In various embodiments, the PPDCs possess one or more of the followinggrowth features (1) they require L-valine for growth in culture; (2)they are capable of growth in atmospheres containing oxygen from about5% to at least about 20% (3) they have the potential for at least about40 doublings in culture before reaching senescence; and (4) they attachand expand on a coated or uncoated tissue culture vessel, wherein thecoated tissue culture vessel comprises a coating of gelatin, laminin,collagen, polyomithine, vitronectin or fibronectin.

In certain embodiments the PPDCs possess a normal karyotype, which ismaintained as the cells are passaged. Karyotyping is particularly usefulfor identifying and distinguishing neonatal from maternal cells derivedfrom placenta. Methods for karyotyping are available and known to thoseof skill in the art.

In other embodiments, the PPDCs may be characterized by production ofcertain proteins, including (1) production of at least one of tissuefactor, vimentin, and alpha-smooth muscle actin; and (2) production ofat least one of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 andHLA-A, B, C cell surface markers, as detected by flow cytometry. Inother embodiments, the PPDCs may be characterized by lack of productionof at least one of CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD 178,B7-H2, HLA-G, and HLA-DR, DP, DQ cell surface markers, as detected byflow cytometry. Particularly preferred are cells that produce at leasttwo of tissue factor, vimentin, and alpha-smooth muscle actin. Morepreferred are those cells producing all three of the proteins tissuefactor, vimentin, and alpha-smooth muscle actin.

In other embodiments, the PPDCs may be characterized by gene expression,which relative to a human cell that is a fibroblast, a mesenchymal stemcell, or an ileac crest bone marrow cell, is increased for a geneencoding at least one of interleukin 8; reticulon 1; chemokine (C--X--Cmotif) ligand 1 (melonoma growth stimulating activity, alpha); chemokine(C--X--C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine(C--X--C motif) ligand 3; tumor necrosis factor, alpha-induced protein3; C-type lectin superfamily member 2; Wilms tumor 1; aldehydedehydrogenase 1 family member A2; renin; oxidized low densitylipoprotein receptor 1; Homo sapiens clone IMAGE:4179671; protein kinaseC zeta; hypothetical protein DKFZp564F013; downregulated in ovariancancer 1; and Homo sapiens gene from clone DKFZp547k1113.

In yet other embodiments, the PPDCs may be characterized by geneexpression, which relative to a human cell that is a fibroblast, amesenchymal stem cell, or an ileac crest bone marrow cell, is reducedfor a gene encoding at least one of: short stature homeobox 2; heatshock 27 kDa protein 2; chemokine (C--X--C motif) ligand 12 (stromalcell-derived factor 1); elastin (supravalvular aortic stenosis,Williams-Beuren syndrome); Homo sapiens mRNA; cDNA DKFZp586M2022 (fromclone DKFZp586M2022); mesenchyme homeo box 2 (growth arrest-specifichomeo box); sine oculis homeobox homolog 1 (Drosophila); crystallin,alpha B; disheveled associated activator of morphogenesis 2;DKFZP586B2420 protein; similar to neuralin 1; tetranectin (plasminogenbinding protein); src homology three (SH3) and cysteine rich domain;cholesterol 25-hydroxylase; runt-related transcription factor 3;interleukin 11 receptor, alpha; procollagen C-endopeptidase enhancer;frizzled homolog 7 (Drosophila); hypothetical gene BC008967; collagen,type VIII, alpha 1; tenascin C (hexabrachion); iroquois homeobox protein5; hephaestin; integrin, beta 8; synaptic vesicle glycoprotein 2;neuroblastoma, suppression of tumorigenicity 1; insulin-like growthfactor binding protein 2, 36 kDa; Homo sapiens cDNA FLJ12280 fis, cloneMAMMA1001744; cytokine receptor-like factor 1; potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 4; integrin, beta 7; transcriptional co-activator withPDZ-binding motif (TAZ); sine oculis homeobox homolog 2 (Drosophila);KIAA1034 protein; vesicle-associated membrane protein 5 (myobrevin);EGF-containing fibulin-like extracellular matrix protein 1; early growthresponse 3; distal-less homeo box 5; hypothetical protein FLJ20373;aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroiddehydrogenase, type II); biglycan; transcriptional co-activator withPDZ-binding motif (TAZ); fibronectin 1; proenkephalin; integrin,beta-like 1 (with EGF-like repeat domains); Homo sapiens mRNA fulllength insert cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein;natriuretic peptide receptor C/guanylate cyclase C (atrionatriureticpeptide receptor C); hypothetical protein FLJ14054; Homo sapiens mRNA;cDNA DKFZp564B222 (from clone DKFZp564B222); BCL2/adenovirus E1B 19 kDainteracting protein 3-like; AE binding protein 1; and cytochrome coxidase subunit VIIa polypeptide 1 (muscle).

In other embodiments, the PPDCs may be characterized by secretion of atleast one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO,MIP1a, RANTES, and TIMP1. In alternative embodiments, the PPDCs may becharacterized by lack of secretion of at least one of TGF-beta2, ANG2,PDGFbb, MIP1b, 1309, MDC, and VEGF, as detected by ELISA.

In preferred embodiments, the cell comprises two or more of theabove-listed growth, protein/surface marker production, gene expressionor substance-secretion characteristics. More preferred are those cellscomprising, three, four, or five or more of the characteristics. Stillmore preferred are PPDCs comprising six, seven, or eight or more of thecharacteristics. Still more preferred presently are those cellscomprising all of above characteristics.

Among cells that are presently preferred for use with the invention inseveral of its aspects are postpartum cells having the characteristicsdescribed above and more particularly those wherein the cells havenormal karyotypes and maintain normal karyotypes with passaging, andfurther wherein the cells express each of the markers CD10, CD13, CD44,CD73, CD90, PDGFr-alpha, and HLA-A, B, C, wherein the cells produce theimmunologically-detectable proteins which correspond to the listedmarkers. Still more preferred are those cells which in addition to theforegoing do not produce proteins corresponding to any of the markersCD31, CD34, CD45, CD117, CD141, or HLA-DR, DP, DQ, as detected by flowcytometry.

Certain cells having the potential to differentiate along lines leadingto various phenotypes are unstable and thus can spontaneouslydifferentiate. Presently preferred for use with the invention are cellsthat do not spontaneously differentiate, for example along neural lines.Preferred cells, when grown in Growth Medium, are substantially stablewith respect to the cell markers produced on their surface, and withrespect to the expression pattern of various genes, for example asdetermined using an Affymetrix GENECHIP. The cells remain substantiallyconstant, for example in their surface marker characteristics overpassaging, through multiple population doublings.

However, one feature of PPDCs is that they may be deliberately inducedto differentiate into various lineage phenotypes by subjecting them todifferentiation-inducing cell culture conditions. Of use in treatment ofcertain ocular degenerative conditions, the PPDCs may be induced todifferentiate into neural phenotypes using one or more methods known inthe art. For instance, as exemplified herein, PPDCs may be plated onflasks coated with laminin in Neurobasal-A medium (Invitrogen, Carlsbad,Calif.) containing B27 (B27 supplement, Invitrogen), L-glutamine andPenicillin/Streptomycin, the combination of which is referred to hereinas Neural Progenitor Expansion (NPE) medium. NPE media may be furthersupplemented with bFGF and/or EGF. Alternatively, PPDCs may be inducedto differentiate in vitro by (1) co-culturing the PPDCs with neuralprogenitor cells, or (2) growing the PPDCs in neural progenitorcell-conditioned medium.

Differentiation of the PPDCs into neural phenotypes may be demonstratedby a bipolar cell morphology with extended processes. The induced cellpopulations may stain positive for the presence of nestin.Differentiated PPDCs may be assessed by detection of nestin, TuJ1 (BIIItubulin), GFAP, tyrosine hydroxylase, GABA, 04 and/or MBP. In someembodiments, PPDCs have exhibited the ability to form three-dimensionalbodies characteristic of neuronal stem cell formation of neurospheres.

Cell Populations, Modifications, Components and Products

Another aspect of the invention features populations of cells isolatedfrom placental or umbilical tissue. In a preferred embodiment, thepopulations comprise the PPDCs described above, and these cellpopulations are described in the section below. In some embodiments, thecell population is heterogeneous. A heterogeneous cell population of theinvention may comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95% PPDCs. The heterogeneous cell populations of theinvention may further comprise stem cells or other progenitor cells,such as epithelial or neural progenitor cells, or it may furthercomprise fully differentiated cells. In some embodiments, the populationis substantially homogeneous, i.e., comprises substantially only PPDCs(preferably at least about 96%, 97%, 98%, 99% or more PPDCs). Thehomogeneous cell population of the invention may comprise umbilicus- orplacenta-derived cells. Homogeneous populations of umbilicus-derivedcells are preferably free of cells of maternal lineage. Homogeneouspopulations of placenta-derived cells may be of neonatal or maternallineage. Homogeneity of a cell population may be achieved by any methodknown in the art, for example, by cell sorting (e.g., flow cytometry) orby clonal expansion in accordance with known methods. Thus, preferredhomogeneous PPDC populations may comprise a clonal cell line ofpostpartum-derived cells. Such populations are particularly useful whena cell clone with highly desirable functionality has been isolated.

Also provided herein are populations of cells incubated in the presenceof one or more factors, or under conditions, that stimulate stem celldifferentiation along a desired pathway (e.g., neural, epithelial). Suchfactors are known in the art and the skilled artisan will appreciatethat determination of suitable conditions for differentiation can beaccomplished with routine experimentation. Optimization of suchconditions can be accomplished by statistical experimental design andanalysis, for example response surface methodology allows simultaneousoptimization of multiple variables, for example in a biological culture.Presently preferred factors include, but are not limited to factors,such as growth or trophic factors, demethylating agents, co-culture withneural or epithelial lineage cells or culture in neural or epitheliallineage cell-conditioned medium, as well other conditions known in theart to stimulate stem cell differentiation along these pathways (forfactors useful in neural differentiation, see, e.g., Lang, K. J. D. etal., 2004, J. Neurosci. Res. 76: 184-192; Johe, K. K. et al., 1996,Genes Devel. 10: 3129-3140; Gottleib, D., 2002, Ann. Rev. Neurosci. 25:381-407).

Postpartum cells, preferably PPDCs, may also be genetically modified toproduce therapeutically useful gene products, or to produceantineoplastic agents for treatment of tumors, for example. Geneticmodification may be accomplished using any of a variety of vectorsincluding, but not limited to, integrating viral vectors, e.g.,retrovirus vector or adeno-associated viral vectors; non-integratingreplicating vectors, e.g., papilloma virus vectors, SV40 vectors,adenoviral vectors; or replication-defective viral vectors. Othermethods of introducing DNA into cells include the use of liposomes,electroporation, a particle gun, or by direct DNA injection.

Hosts cells are preferably transformed or transfected with DNAcontrolled by or in operative association with, one or more appropriateexpression control elements such as promoter or enhancer sequences,transcription terminators, polyadenylation sites, among others, and aselectable marker. Any promoter may be used to drive the expression ofthe inserted gene. For example, viral promoters include, but are notlimited to, the CMV promoter/enhancer, SV 40, papillomavirus,Epstein-Barr virus or elastin gene promoter. In some embodiments, thecontrol elements used to control expression of the gene of interest canallow for the regulated expression of the gene so that the product issynthesized only when needed in vivo. If transient expression isdesired, constitutive promoters are preferably used in a non-integratingand/or replication-defective vector. Alternatively, inducible promoterscould be used to drive the expression of the inserted gene whennecessary. Inducible promoters include, but are not limited to thoseassociated with metallothionein and heat shock proteins.

Following the introduction of the foreign DNA, engineered cells may beallowed to grow in enriched media and then switched to selective media.The selectable marker in the foreign DNA confers resistance to theselection and allows cells to stably integrate the foreign DNA as, forexample, on a plasmid, into their chromosomes and grow to form fociwhich, in turn, can be cloned and expanded into cell lines. This methodcan be advantageously used to engineer cell lines that express the geneproduct.

Cells may be genetically engineered to “knock out” or “knock down”expression of factors that promote inflammation or rejection at theimplant site. Negative modulatory techniques for the reduction of targetgene expression levels or target gene product activity levels arediscussed below. “Negative modulation,” as used herein, refers to areduction in the level and/or activity of target gene product relativeto the level and/or activity of the target gene product in the absenceof the modulatory treatment. The expression of a gene native to a neuronor glial cell can be reduced or knocked out using a number of techniquesincluding, for example, inhibition of expression by inactivating thegene using the homologous recombination technique. Typically, an exonencoding an important region of the protein (or an exon 5′ to thatregion) is interrupted by a positive selectable marker, e.g., neo,preventing the production of normal mRNA from the target gene andresulting in inactivation of the gene. A gene may also be inactivated bycreating a deletion in part of a gene, or by deleting the entire gene.By using a construct with two regions of homology to the target genethat are far apart in the genome, the sequences intervening the tworegions can be deleted (Mombaerts et al., 1991, Proc. Nat. Acad. Sci.U.S.A. 88:3084). Antisense, DNAzymes, ribozymes, small interfering RNA(siRNA) and other such molecules that inhibit expression of the targetgene can also be used to reduce the level of target gene activity. Forexample, antisense RNA molecules that inhibit the expression of majorhistocompatibility gene complexes (HLA) have been shown to be mostversatile with respect to immune responses. Still further, triple helixmolecules can be utilized in reducing the level of target gene activity.These techniques are described in detail by L. G. Davis et al. (eds),1994, BASIC METHODS IN MOLECULAR BIOLOGY, 2nd ed., Appleton & Lange,Norwalk, Conn.

In other aspects, the invention provides cell lysates and cell solublefractions prepared from postpartum stem cells, preferably PPDCs, orheterogeneous or homogeneous cell populations comprising PPDCs, as wellas PPDCs or populations thereof that have been genetically modified orthat have been stimulated to differentiate along a neurogenic pathway.Such lysates and fractions thereof have many utilities. Use of the celllysate soluble fraction (i.e., substantially free of membranes) in vivo,for example, allows the beneficial intracellular milieu to be usedallogeneically in a patient without introducing an appreciable amount ofthe cell surface proteins most likely to trigger rejection, or otheradverse immunological responses. Methods of lysing cells are well knownin the art and include various means of mechanical disruption, enzymaticdisruption, or chemical disruption, or combinations thereof. Such celllysates may be prepared from cells directly in their Growth Medium andthus containing secreted growth factors and the like, or may be preparedfrom cells washed free of medium in, for example, PBS or other solution.Washed cells may be resuspended at concentrations greater than theoriginal population density if preferred.

In one embodiment, whole cell lysates are prepared, e.g., by disruptingcells without subsequent separation of cell fractions. In anotherembodiment, a cell membrane fraction is separated from a solublefraction of the cells by routine methods known in the art, e.g.,centrifugation, filtration, or similar methods.

Cell lysates or cell soluble fractions prepared from populations ofpostpartum-derived cells may be used as is, further concentrated, by forexample, ultrafiltration or lyophilization, or even dried, partiallypurified, combined with pharmaceutically-acceptable carriers or diluentsas are known in the art, or combined with other compounds such asbiologicals, for example pharmaceutically useful protein compositions.Cell lysates or fractions thereof may be used in vitro or in vivo, aloneor for example, with autologous or syngeneic live cells. The lysates, ifintroduced in vivo, may be introduced locally at a site of treatment, orremotely to provide, for example needed cellular growth factors to apatient.

In a further embodiment, postpartum cells, preferably PPDCs, can becultured in vitro to produce biological products in high yield. Forexample, such cells, which either naturally produce a particularbiological product of interest (e.g., a trophic factor), or have beengenetically engineered to produce a biological product, can be clonallyexpanded using the culture techniques described herein. Alternatively,cells may be expanded in a medium that induces differentiation to adesired lineage. In either case, biological products produced by thecell and secreted into the medium can be readily isolated from theconditioned medium using standard separation techniques, e.g., such asdifferential protein precipitation, ion-exchange chromatography, gelfiltration chromatography, electrophoresis, and HPLC, to name a few. A“bioreactor” may be used to take advantage of the flow method forfeeding, for example, a three-dimensional culture in vitro. Essentially,as fresh media is passed through the three-dimensional culture, thebiological product is washed out of the culture and may then be isolatedfrom the outflow, as above.

Alternatively, a biological product of interest may remain within thecell and, thus, its collection may require that the cells be lysed, asdescribed above. The biological product may then be purified using anyone or more of the above-listed techniques.

In other embodiments, the invention provides conditioned medium fromcultured postpartum cells, preferably PPDCs, for use in vitro and invivo as described below. Use of such conditioned medium allows thebeneficial trophic factors secreted by the postpartum cells to be usedallogeneically in a patient without introducing intact cells that couldtrigger rejection, or other adverse immunological responses. Conditionedmedium is prepared by culturing cells in a culture medium, then removingthe cells from the medium.

Conditioned medium prepared from populations of postpartum-derived cellsmay be used as is, further concentrated, by for example, ultrafiltrationor lyophilization, or even dried, partially purified, combined withpharmaceutically-acceptable carriers or diluents as are known in theart, or combined with other compounds such as biologicals, for examplepharmaceutically useful protein compositions. Conditioned medium may beused in vitro or in vivo, alone or for example, with autologous orsyngeneic live cells. The conditioned medium, if introduced in vivo, maybe introduced locally at a site of treatment, or remotely to provide,for example needed cellular growth or trophic factors to a patient.

In another embodiment, an extracellular matrix (ECM) produced byculturing postpartum cells (preferably PPDCs) on liquid, solid orsemi-solid substrates is prepared, collected and utilized as analternative to implanting live cells into a subject in need of tissuerepair or replacement. The cells are cultured in vitro, on a threedimensional framework as described elsewhere herein, under conditionssuch that a desired amount of ECM is secreted onto the framework. Thecells and the framework are removed, and the ECM processed for furtheruse, for example, as an injectable preparation. To accomplish this,cells on the framework are killed and any cellular debris removed fromthe framework. This process may be carried out in a number of differentways. For example, the living tissue can be flash-frozen in liquidnitrogen without a cryopreservative, or the tissue can be immersed insterile distilled water so that the cells burst in response to osmoticpressure.

Once the cells have been killed, the cellular membranes may be disruptedand cellular debris removed by treatment with a mild detergent rinse,such as EDTA, CHAPS or a zwitterionic detergent. Alternatively, thetissue can be enzymatically digested and/or extracted with reagents thatbreak down cellular membranes and allow removal of cell contents.Example of such enzymes include, but are not limited to, hyaluronidase,dispase, proteases, and nucleases. Examples of detergents includenon-ionic detergents such as, for example, alkylaryl polyether alcohol(TRITON X-100), octylphenoxy polyethoxy-ethanol (Rohm and HaasPhiladelphia, Pa.), BRIJ-35, a polyethoxyethanol lauryl ether (AtlasChemical Co., San Diego, Calif.), polysorbate 20 (TWEEN 20), apolyethoxyethanol sorbitan monolaureate (Rohm and Haas), polyethylenelauryl ether (Rohm and Haas); and ionic detergents such as, for example,sodium dodecyl sulphate, sulfated higher aliphatic alcohols, sulfonatedalkanes and sulfonated alkylarenes containing 7 to 22 carbon atoms in abranched or unbranched chain.

The collection of the ECM can be accomplished in a variety of ways,depending, for example, on whether the new tissue has been formed on athree-dimensional framework that is biodegradable or non-biodegradable.For example, if the framework is non-biodegradable, the ECM can beremoved by subjecting the framework to sonication, high-pressure waterjets, mechanical scraping, or mild treatment with detergents or enzymes,or any combination of the above.

If the framework is biodegradable, the ECM can be collected, forexample, by allowing the framework to degrade or dissolve in solution.Alternatively, if the biodegradable framework is composed of a materialthat can itself be injected along with the ECM, the framework and theECM can be processed in toto for subsequent injection. Alternatively,the ECM can be removed from the biodegradable framework by any of themethods described above for collection of ECM from a non-biodegradableframework. All collection processes are preferably designed so as not todenature the ECM.

After it has been collected, the ECM may be processed further. Forexample, the ECM can be homogenized to fine particles using techniqueswell known in the art such as by sonication, so that it can pass througha surgical needle. The components of the ECM can be crosslinked, ifdesired, by gamma irradiation. Preferably, the ECM can be irradiatedbetween 0.25 to 2 mega rads to sterilize and cross link the ECM.Chemical crosslinking using agents that are toxic, such asglutaraldehyde, is possible but not generally preferred.

The amounts and/or ratios of proteins, such as the various types ofcollagen present in the ECM, may be adjusted by mixing the ECM producedby the cells of the invention with ECM of one or more other cell types.In addition, biologically active substances such as proteins, growthfactors and/or drugs, can be incorporated into the ECM. Exemplarybiologically active substances include tissue growth factors, such asTGF-beta, and the like, which promote healing and tissue repair at thesite of the injection. Such additional agents may be utilized in any ofthe embodiments described herein above, e.g., with whole cell lysates,soluble cell fractions, or further purified components and productsproduced by the cells.

In another embodiment, postpartum cells (preferably PPDCs) are used toresurface Bruch's membrane that has undergone modifications due to age.Such modifications include, for example, increased thickness, depositionof extracellular matrix and lipids, cross-linking of protein,non-enzymatic formation of advanced glycation end products. In oneembodiment, the age-related changes in Bruch's membrane causedissociation of RPE cells from the Bruch's membrane, and ultimatelyresult in photoreceptor cell death. In one aspect of the presentinvention, the resurfacing of Bruch's membrane by postpartum cells(preferably PPDCs) prevents the dissociation of RPE cells from theBruch's membrane. In an alternate embodiment, the postpartum cellspromote the attachment of RPE cells to Bruch's membrane. The RPE cellsmay be endogenous, or they may be transplanted RPE cells.

Pharmaceutical Compositions

In another aspect, the invention provides pharmaceutical compositionsthat utilize postpartum cells, preferably PPDCs, cell populations andcell components and products in various methods for treatment of oculardegenerative conditions. Certain embodiments encompass pharmaceuticalcompositions comprising live cells (e.g., PPDCs alone or admixed withother cell types). Other embodiments encompass pharmaceuticalcompositions comprising PPDC cellular components (e.g., cell lysates,soluble cell fractions, conditioned medium, ECM, or components of any ofthe foregoing) or products (e.g., trophic and other biological factorsproduced naturally by PPDCs or through genetic modification, conditionedmedium from PPDC culture). In either case, the pharmaceuticalcomposition may further comprise other active agents, such asanti-inflammatory agents, anti-apoptotic agents, antioxidants, growthfactors, neurotrophic factors or neuroregenerative, neuroprotective orophthalmic drugs as known in the art.

Examples of other components that may be added to PPDC pharmaceuticalcompositions include, but are not limited to: (1) other neuroprotectiveor neurobeneficial drugs; (2) selected extracellular matrix components,such as one or more types of collagen known in the art, and/or growthfactors, platelet-rich plasma, and drugs (alternatively, PPDCs may begenetically engineered to express and produce growth factors); (3)anti-apoptotic agents (e.g., erythropoietin (EPO), EPO mimetibody,thrombopoietin, insulin-like growth factor (IGF)-I, IGF-II, hepatocytegrowth factor, caspase inhibitors); (4) anti-inflammatory compounds(e.g., p38 MAP kinase inhibitors, TGF-beta inhibitors, statins, IL-6 andIL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, andnon-steroidal anti-inflammatory drugs (NSAIDS) (such as TEPDXALIN,TOLMETIN, and SUPROFEN); (5) immunosuppressive or immunomodulatoryagents, such as calcineurin inhibitors, mTOR inhibitors,antiproliferatives, corticosteroids and various antibodies; (6)antioxidants such as probucol, vitamins C and E, conenzyme Q-10,glutathione, L-cysteine and N-acetylcysteine; and (6) local anesthetics,to name a few.

Pharmaceutical compositions of the invention comprise postpartum cells(preferably PPDCs), or components or products thereof, formulated with apharmaceutically acceptable carrier or medium. Suitable pharmaceuticallyacceptable carriers include water, salt solution (such as Ringer'ssolution), alcohols, oils, gelatins, and carbohydrates, such as lactose,amylose, or starch, fatty acid esters, hydroxymethylcellulose, andpolyvinyl pyrolidine. Such preparations can be sterilized, and ifdesired, mixed with auxiliary agents such as lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, and coloring. Typically, but not exclusively,pharmaceutical compositions comprising cellular components or products,but not live cells, are formulated as liquids. Pharmaceuticalcompositions comprising PPDC live cells are typically formulated asliquids, semisolids (e.g., gels) or solids (e.g., matrices, scaffoldsand the like, as appropriate for ophthalmic tissue engineering).

Pharmaceutical compositions may comprise auxiliary components as wouldbe familiar to medicinal chemists or biologists. For example, they maycontain antioxidants in ranges that vary depending on the kind ofantioxidant used. Reasonable ranges for commonly used antioxidants areabout 0.01% to about 0.15% weight by volume of EDTA, about 0.01% toabout 2.0% weight volume of sodium sulfite, and about 0.01% to about2.0% weight by volume of sodium metabisulfite. One skilled in the artmay use a concentration of about 0.1% weight by volume for each of theabove. Other representative compounds include mercaptopropionyl glycine,N-acetyl cysteine, beta-mercaptoethylamine, glutathione and similarspecies, although other antioxidant agents suitable for ocularadministration, e.g. ascorbic acid and its salts or sulfite or sodiummetabisulfite may also be employed.

A buffering agent may be used to maintain the pH of eye dropformulations in the range of about 4.0 to about 8.0; so as to minimizeirritation of the eye. For direct intravitreal or intraocular injection,formulations should be at pH 7.2 to 7.5, preferably at pH 7.3-7.4. Theophthalmologic compositions may also include tonicity agents suitablefor administration to the eye. Among those suitable is sodium chlorideto make formulations approximately isotonic with 0.9% saline solution.

In certain embodiments, pharmaceutical compositions are formulated withviscosity enhancing agents. Exemplary agents are hydroxyethylcellulose,hydroxypropylcellulose, methylcellulose, and polyvinylpyrrolidone. Thepharmaceutical compositions may have cosolvents added if needed.Suitable cosolvents may include glycerin, polyethylene glycol (PEG),polysorbate, propylene glycol, and polyvinyl alcohol. Preservatives mayalso be included, e.g., benzalkonium chloride, benzethonium chloride,chlorobutanol, phenylmercuric acetate or nitrate, thimerosal, or methylor propylparabens.

Formulations for injection are preferably designed for single-useadministration and do not contain preservatives. Injectable solutionsshould have isotonicity equivalent to 0.9% sodium chloride solution(osmolality of 290-300 milliosmoles). This may be attained by additionof sodium chloride or other co-solvents as listed above, or excipientssuch as buffering agents and antioxidants, as listed above.

The tissues of the anterior chamber of the eye are bathed by the aqueoushumor, while the retina is under continuous exposure to the vitreous.These fluids/gels exist in a highly reducing redox state because theycontain antioxidant compounds and enzymes. Therefore, it may beadvantageous to include a reducing agent in the ophthalmologiccompositions. Suitable reducing agents include N-acetylcysteine,ascorbic acid or a salt form, and sodium sulfite or metabisulfite, withascorbic acid and/or N-acetylcysteine or glutathione being particularlysuitable for injectable solutions.

Pharmaceutical compositions comprising cells, cell components or cellproducts may be delivered to the eye of a patient in one or more ofseveral delivery modes known in the art. In one embodiment that may besuitable for use in some instances, the compositions are topicallydelivered to the eye in eye drops or washes. In another embodiment, thecompositions may be delivered to various locations within the eye viaperiodic intraocular injection or by infusion in an irrigating solutionsuch as BSS or BSS PLUS (Alcon USA, Fort Worth, Tex.). Alternatively,the compositions may be applied in other ophthalmologic dosage formsknown to those skilled in the art, such as pre-formed or in situ-formedgels or liposomes, for example as disclosed in U.S. Pat. No. 5,718,922to Herrero-Vanrell. In another embodiment, the composition may bedelivered to or through the lens of an eye in need of treatment via acontact lens (e.g. Lidofilcon B, Bausch & Lomb CW79 or DELTACON(Deltafilcon A) or other object temporarily resident upon the surface ofthe eye. In other embodiments, supports such as a collagen cornealshield (e.g. BIO-COR dissolvable corneal shields, Summit Technology,Watertown, Mass.) can be employed. The compositions can also beadministered by infusion into the eyeball, either through a cannula froman osmotic pump (ALZET, Alza Corp., Palo Alto, Calif.) or byimplantation of timed-release capsules (OCCUSENT) or biodegradable disks(OCULEX, OCUSERT). These routes of administration have the advantage ofproviding a continuous supply of the pharmaceutical composition to theeye. This may be an advantage for local delivery to the cornea, forexample.

Pharmaceutical compositions comprising live cells in a semi-solid orsolid carrier are typically formulated for surgical implantation at thesite of ocular damage or distress. It will be appreciated that liquidcompositions also may be administered by surgical procedures. Inparticular embodiments, semi-solid or solid pharmaceutical compositionsmay comprise semi-permeable gels, lattices, cellular scaffolds and thelike, which may be non-biodegradable or biodegradable. For example, incertain embodiments, it may be desirable or appropriate to sequester theexogenous cells from their surroundings, yet enable the cells to secreteand deliver biological molecules to surrounding cells. In theseembodiments, cells may be formulated as autonomous implants comprisingliving PPDCs or cell population comprising PPDCs surrounded by anon-degradable, selectively permeable barrier that physically separatesthe transplanted cells from host tissue. Such implants are sometimesreferred to as “immunoprotective,” as they have the capacity to preventimmune cells and macromolecules from killing the transplanted cells inthe absence of pharmacologically induced immunosuppression (for a reviewof such devices and methods, see, e.g., P. A. Tresco et al., 2000, Adv.Drug Delivery Rev. 42: 3-27).

In other embodiments, different varieties of degradable gels andnetworks are utilized for the pharmaceutical compositions of theinvention. For example, degradable materials particularly suitable forsustained release formulations include biocompatible polymers, such aspoly (lactic acid), poly (lactic-co-glycolic acid), methylcellulose,hyaluronic acid, collagen, and the like. The structure, selection anduse of degradable polymers in drug delivery vehicles have been reviewedin several publications, including, A. Domb et al., 1992, Polymers forAdvanced Technologies 3:279. U.S. Pat. No. 5,869,079 to Wong et al.disclose combinations of hydrophilic and hydrophobic entities in abiodegradable sustained release ocular implant. In addition, U.S. Pat.No. 6,375,972 to Guo et al., U.S. Pat. No. 5,902,598 to Chen et al.,U.S. Pat. No. 6,331,313 to Wong et al., U.S. Pat. No. 5,707,643 to Oguraet al., U.S. Pat. No. 5,466,233 to Weiner et al. and U.S. Pat. No.6,251,090 to Avery et al. each describes intraocular implant devices andsystems that may be used to deliver pharmaceutical compositions.

In other embodiments, e.g., for repair of neural lesions, such as adamaged or severed optic nerve, it may be desirable or appropriate todeliver the cells on or in a biodegradable, preferably bioresorbable orbioabsorbable, scaffold or matrix. These typically three-dimensionalbiomaterials contain the living cells attached to the scaffold,dispersed within the scaffold, or incorporated in an extracellularmatrix entrapped in the scaffold. Once implanted into the target regionof the body, these implants become integrated with the host tissue,wherein the transplanted cells gradually become established (see, e.g.,P. A. Tresco et al., 2000, supra; see also D. W. Hutmacher, 2001, J.Biomater. Sci. Polymer Edn. 12: 107-174).

Examples of scaffold or matrix (sometimes referred to collectively as“framework”) material that may be used in the present invention includenonwoven mats, porous foams, or self-assembling peptides. Nonwoven matsmay, for example, be formed using fibers comprised of a syntheticabsorbable copolymer of glycolic and lactic acids (PGA/PLA), sold underthe trade name VICRYL (Ethicon, Inc., Somerville, N.J.), Foams, composedof, for example, poly (epsilon-caprolactone)/poly (glycolic acid)(PCL/PGA) copolymer, formed by processes such as freeze-drying, orlyophilized, as discussed in U.S. Pat. No. 6,355,699 also may beutilized. Hydrogels such as self-assembling peptides (e.g., RAD16) mayalso be used. In situ-forming degradable networks are also suitable foruse in the invention (see, e.g., Anseth, K. S. et al., 2002, J.Controlled Release 78: 199-209; Wang, D. et al., 2003, Biomaterials 24:3969-3980; U.S. Patent Publication 2002/0022676 to He et al.). Thesematerials are formulated as fluids suitable for injection, and then maybe induced by a variety of means (e.g., change in temperature, pH,exposure to light) to form degradable hydrogel networks in situ or invivo.

In another embodiment, the framework is a felt, which can be composed ofa multifilament yarn made from a bioabsorbable material, e.g., PGA, PLA,PCL copolymers or blends, or hyaluronic acid. The yarn is made into afelt using standard textile processing techniques consisting ofcrimping, cutting, carding and needling. In another embodiment, cellsare seeded onto foam scaffolds that may be composite structures.

In many of the abovementioned embodiments, the framework may be moldedinto a useful shape. Furthermore, it will be appreciated that PPDCs maybe cultured on pre-formed, non-degradable surgical or implantabledevices, e.g., in a manner corresponding to that used for preparingfibroblast-containing GDC endovascular coils, for instance (Marx, W. F.et al., 2001, Am. J. Neuroradiol. 22: 323-333).

The matrix, scaffold or device may be treated prior to inoculation ofcells in order to enhance cell attachment. For example, prior toinoculation, nylon matrices can be treated with 0.1 molar acetic acidand incubated in polylysine, PBS, and/or collagen to coat the nylon.Polystyrene can be similarly treated using sulfuric acid. The externalsurfaces of a framework may also be modified to improve the attachmentor growth of cells and differentiation of tissue, such as by plasmacoating the framework or addition of one or more proteins (e.g.,collagens, elastic fibers, reticular fibers), glycoproteins,glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate,chondroitin-6-sulfate, dermatan sulfate, keratin sulfate), a cellularmatrix, and/or other materials such as, but not limited to, gelatin,alginates, agar, agarose, and plant gums, among others.

Frameworks containing living cells are prepared according to methodsknown in the art. For example, cells can be grown freely in a culturevessel to sub-confluency or confluency, lifted from the culture andinoculated onto the framework. Growth factors may be added to theculture medium prior to, during, or subsequent to inoculation of thecells to trigger differentiation and tissue formation, if desired.Alternatively, the frameworks themselves may be modified so that thegrowth of cells thereon is enhanced, or so that the risk of rejection ofthe implant is reduced. Thus, one or more biologically active compounds,including, but not limited to, anti-inflammatory agents,immunosuppressants or growth factors, may be added to the framework forlocal release.

Methods of Use

Postpartum cells, preferably PPDCs, or cell populations, components ofor products produced by such cells, may be used in a variety of ways tosupport and facilitate repair and regeneration of ocular cells andtissues. Such utilities encompass in vitro, ex vivo and in vivo methods.The methods set forth below are directed to PPDCs, but other postpartumcells may also be suitable for use in those methods.

In Vitro and Ex Vivo Methods

In one embodiment, PPDCs may be used in vitro to screen a wide varietyof compounds for effectiveness and cytotoxicity of pharmaceuticalagents, growth factors, regulatory factors, and the like. For example,such screening may be performed on substantially homogeneous populationsof PPDCs to assess the efficacy or toxicity of candidate compounds to beformulated with, or co-administered with, the PPDCs, for treatment of aan ocular condition. Alternatively, such screening may be performed onPPDCs that have been stimulated to differentiate into a cell type foundin the eye, or progenitor thereof, for the purpose of evaluating theefficacy of new pharmaceutical drug candidates. In this embodiment, thePPDCs are maintained in vitro and exposed to the compound to be tested.The activity of a potentially cytotoxic compound can be measured by itsability to damage or kill cells in culture. This may readily be assessedby vital staining techniques. The effect of growth or regulatory factorsmay be assessed by analyzing the number or robustness of the culturedcells, as compared with cells not exposed to the factors. This may beaccomplished using standard cytological and/or histological techniques,including the use of immunocytochemical techniques employing antibodiesthat define type-specific cellular antigens.

In a further embodiment, as discussed above, PPDCs can be cultured invitro to produce biological products that are either naturally producedby the cells, or produced by the cells when induced to differentiateinto other lineages, or produced by the cells via genetic modification.For instance, TIMP1, TPO, KGF, HGF, FGF, HBEGF, BDNF, MIP1b, MCP1,RANTES, 1309, TARC, MDC, and IL-8 were found to be secreted fromumbilicus-derived cells grown in Growth Medium. TIMP1, TPO, KGF, HGF,HBEGF, BDNF, MIP1a, MCP-1, RANTES, TARC, Eotaxin, and IL-8 were found tobe secreted from placenta-derived PPDCs cultured in Growth Medium (seeExamples). Some of these trophic factors, such as BDNF and IL-6, haveimportant roles in neural regeneration. Other trophic factors, as yetundetected or unexamined, of use in repair and regeneration of oculartissues, are likely to be produced by PPDCs and possibly secreted intothe medium.

In this regard, another embodiment of the invention features use ofPPDCs for production of conditioned medium, either from undifferentiatedPPDCs or from PPDCs incubated under conditions that stimulatedifferentiation. Such conditioned media are contemplated for use in invitro or ex vivo culture of epithelial or neural precursor cells, forexample, or in vivo to support transplanted cells comprising homogeneouspopulations of PPDCs or heterogeneous populations comprising PPDCs andother progenitors, for example.

Yet another embodiment comprises the use of PPCD cell lysates, solublecell fractions or components thereof, or ECM or components thereof, fora variety of purposes. As mentioned above, some of these components maybe used in pharmaceutical compositions. In other embodiments, a celllysate or ECM is used to coat or otherwise treat substances or devicesto be used surgically, or for implantation, or for ex vivo purposes, topromote healing or survival of cells or tissues contacted in the courseof such treatments.

As described in Examples 13 and 15, PPDCs have demonstrated the abilityto support survival, growth and differentiation of adult neuralprogenitor cells when grown in co-culture with those cells. Likewise,the experimental results set forth in Example 18 indicates that PPDCsmay function to support cells of the retina via trophic mechanisms.Accordingly, in another embodiment, PPDCs are used advantageously inco-cultures in vitro to provide trophic support to other cells, inparticular neural cells and neural and ocular progenitors (e.g., neuralstem cells and retinal or corneal epithelial stem cells). Forco-culture, it may be desirable for the PPDCs and the desired othercells to be co-cultured under conditions in which the two cell types arein contact. This can be achieved, for example, by seeding the cells as aheterogeneous population of cells in culture medium or onto a suitableculture substrate. Alternatively, the PPDCs can first be grown toconfluence, and then will serve as a substrate for the second desiredcell type in culture. In this latter embodiment, the cells may furtherbe physically separated, e.g., by a membrane or similar device, suchthat the other cell type may be removed and used separately, followingthe co-culture period. Use of PPDCs in co-culture to promote expansionand differentiation of neural or ocular cell types may findapplicability in research and in clinical/therapeutic areas. Forinstance, PPDC co-culture may be utilized to facilitate growth anddifferentiation of such cells in culture, for basic research purposes orfor use in drug screening assays, for example. PPDC co-culture may alsobe utilized for ex vivo expansion of neural or ocular progenitors forlater administration for therapeutic purposes. For example, neural orocular progenitor cells may be harvested from an individual, expanded exvivo in co-culture with PPDCs, then returned to that individual(autologous transfer) or another individual (syngeneic or allogeneictransfer). In these embodiments, it will be appreciated that, followingex vivo expansion, the mixed population of cells comprising the PPDCsand progenitors could be administered to a patient in need of treatment.Alternatively, in situations where autologous transfer is appropriate ordesirable, the co-cultured cell populations may be physically separatedin culture, enabling removal of the autologous progenitors foradministration to the patient.

In Vivo Methods

As set forth in Examples 16, 17, and 18, PPDCs have been shown to beeffectively transplanted into the body, and to supply lost neural orretinal function in animal models accepted for their predictability ofefficacy in humans. These results support a preferred embodiment of theinvention, wherein PPDCs are used in cell therapy for treating an oculardegenerative condition. Once transplanted into a target location in theeye, PPDCs may themselves differentiate into one or more phenotypes, orthey may provide trophic support for ocular cells in situ, or they mayexert a beneficial effect in both of those fashions, among others.

PPDCs may be administered alone (e.g., as substantially homogeneouspopulations) or as admixtures with other cells. As described above,PPDCs may be administered as formulated in a pharmaceutical preparationwith a matrix or scaffold, or with conventional pharmaceuticallyacceptable carriers. Where PPDCs are administered with other cells, theymay be administered simultaneously or sequentially with the other cells(either before or after the other cells). Cells that may be administeredin conjunction with PPDCs include, but are not limited to, neurons,astrocytes, oligodendrocytes, neural progenitor cells, neural stemcells, ocular progenitor cells, retinal or corneal epithelial stem cellsand/or other multipotent or pluripotent stem cells. The cells ofdifferent types may be admixed with the PPDCs immediately or shortlyprior to administration, or they may be co-cultured together for aperiod of time prior to administration.

The PPDCs may be administered with other beneficial drugs, biologicalmolecules, such as growth factors, trophic factors, conditioned medium(from postpartum cells or from progenitor or differentiated cellcultures), or other active agents, such as anti-inflammatory agents,anti-apoptotic agents, antioxidants, growth factors, neurotrophicfactors or neuroregenerative or neuroprotective drugs as known in theart. When PPDCs are administered with other agents, they may beadministered together in a single pharmaceutical composition, or inseparate pharmaceutical compositions, simultaneously or sequentiallywith the other agents (either before or after administration of theother agents).

Examples of other components that may be administered with postpartumcells include, but are not limited to: (1) other neuroprotective orneurobeneficial drugs; (2) selected extracellular matrix components,such as one or more types of collagen known in the art, and/or growthfactors, platelet-rich plasma, and drugs (alternatively, the cells maybe genetically engineered to express and produce growth factors); (3)anti-apoptotic agents (e.g., erythropoietin (EPO), EPO mimetibody,thrombopoietin, insulin-like growth factor (IGF)-I, IGF-II, hepatocytegrowth factor, caspase inhibitors); (4) anti-inflammatory compounds(e.g., p38 MAP kinase inhibitors, TGF-beta inhibitors, statins, IL-6 andIL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, andnon-steroidal anti-inflammatory drugs (NSAIDS) (such as TEPDXALIN,TOLMETIN, and SUPROFEN); (5) immunosuppressive or immunomodulatoryagents, such as calcineurin inhibitors, mTOR inhibitors,antiproliferatives, corticosteroids and various antibodies; (6)antioxidants such as probucol, vitamins C and E, conenzyme Q-10,glutathione, L-cysteine and N-acetylcysteine; and (6) local anesthetics,to name a few.

In one embodiment, PPDCs are administered as undifferentiated cells,i.e., as cultured in Growth Medium. Alternatively, PPDCs may beadministered following exposure in culture to conditions that stimulatedifferentiation toward a desired phenotype.

The cells may be surgically implanted, injected or otherwiseadministered directly or indirectly to the site of ocular damage ordistress. When cells are administered in semi-solid or solid devices,surgical implantation into a precise location in the body is typically asuitable means of administration. Liquid or fluid pharmaceuticalcompositions, however, may be administered to a more general location inthe eye (e.g., topically or intra-ocularly).

Other embodiments encompass methods of treating ocular degenerativeconditions by administering pharmaceutical compositions comprising PPDCcellular components (e.g., cell lysates or components thereof) orproducts (e.g., trophic and other biological factors produced naturallyby PPDCs or through genetic modification, conditioned medium from PPDCculture). Again, these methods may further comprise administering otheractive agents, such as growth factors, neurotrophic factors orneuroregenerative or neuroprotective drugs as known in the art.

Dosage forms and regimes for administering PPDCs or any of the otherpharmaceutical compositions described herein are developed in accordancewith good medical practice, taking into account the condition of theindividual patient, e.g., nature and extent of the ocular degenerativecondition, age, sex, body weight and general medical condition, andother factors known to medical practitioners. Thus, the effective amountof a pharmaceutical composition to be administered to a patient isdetermined by these considerations as known in the art.

It may be desirable or appropriate to pharmacologically immunosuppress apatient prior to initiating cell therapy. This may be accomplishedthrough the use of systemic or local immunosuppressive agents, or it maybe accomplished by delivering the cells in an encapsulated device, asdescribed above. These and other means for reducing or eliminating animmune response to the transplanted cells are known in the art. As analternative, PPDCs may be genetically modified to reduce theirimmunogenicity, as mentioned above.

Survival of transplanted cells in a living patient can be determinedthrough the use of a variety of scanning techniques, e.g., computerizedaxial tomography (CAT or CT) scan, magnetic resonance imaging (MRI) orpositron emission tomography (PET) scans. Determination of transplantsurvival can also be done post mortem by removing the tissue andexamining it visually or through a microscope. Alternatively, cells canbe treated with stains that are specific for neural or ocular cells orproducts thereof, e.g., neurotransmitters. Transplanted cells can alsobe identified by prior incorporation of tracer dyes such as rhodamine-or fluorescein-labeled microspheres, fast blue, ferric microparticles,bisbenzamide or genetically introduced reporter gene products, such asbeta-galactosidase or beta-glucuronidase.

Functional integration of transplanted cells into ocular tissue of asubject can be assessed by examining restoration of the ocular functionthat was damaged or diseased. For example, effectiveness in thetreatment of macular degeneration or other retinopathies may bedetermined by improvement of visual acuity and evaluation forabnormalities and grading of stereoscopic color fundus photographs.(Age-Related Eye Disease Study Research Group, NEI, NIH, AREDS ReportNo. 8, 2001, Arch. Ophthalmol. 119: 1417-1436).

Kits and Banks

In another aspect, the invention provides kits that utilize postpartumcells, preferably PPDCs, cell populations, components and productsthereof in various methods for ocular regeneration and repair asdescribed above. Where used for treatment of ocular degenerativeconditions, or other scheduled treatment, the kits may include one ormore cell populations, including at least postpartum cells and apharmaceutically acceptable carrier (liquid, semi-solid or solid). Thekits also optionally may include a means of administering the cells, forexample by injection. The kits further may include instructions for useof the cells. Kits prepared for field hospital use, such as for militaryuse may include full-procedure supplies including tissue scaffolds,surgical sutures, and the like, where the cells are to be used inconjunction with repair of acute injuries. Kits for assays and in vitromethods as described herein may contain, for example, one or more of (1)PPDCs or components or products of PPDCs, (2) reagents for practicingthe in vitro method, (3) other cells or cell populations, asappropriate, and (4) instructions for conducting the in vitro method.

In yet another aspect, the invention also provides for banking oftissues, cells, cellular components and cell populations of theinvention. As discussed above, the cells are readily cryopreserved. Theinvention therefore provides methods of cryopreserving the cells in abank, wherein the cells are stored frozen and associated with a completecharacterization of the cells based on immunological, biochemical andgenetic properties of the cells. The frozen cells can be thawed andexpanded or used directly for autologous, syngeneic, or allogeneictherapy, depending on the requirements of the procedure and the needs ofthe patient. Preferably, the information on each cryopreserved sample isstored in a computer, which is searchable based on the requirements ofthe surgeon, procedure and patient with suitable matches being madebased on the characterization of the cells or populations. Preferably,the cells of the invention are grown and expanded to the desiredquantity of cells and therapeutic cell compositions are prepared eitherseparately or as co-cultures, in the presence or absence of a matrix orsupport. While for some applications it may be preferable to use cellsfreshly prepared, the remainder can be cryopreserved and banked byfreezing the cells and entering the information in the computer toassociate the computer entry with the samples. Even where it is notnecessary to match a source or donor with a recipient of such cells, forimmunological purposes, the bank system makes it easy to match, forexample, desirable biochemical or genetic properties of the banked cellsto the therapeutic needs. Upon matching of the desired properties with abanked sample, the sample is retrieved and prepared for therapeutic use.Cell lysates, ECM or cellular components prepared as described hereinmay also be cryopreserved or otherwise preserved (e.g., bylyophilization) and banked in accordance with the present invention.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.

As used in the following examples and elsewhere in the specification,the term Growth Medium generally refers to a medium sufficient for theculturing of PPDCs. In particular, one presently preferred medium forthe culturing of the cells of the invention in comprises Dulbecco'sModified Essential Media (also abbreviated DMEM herein). Particularlypreferred is DMEM—low glucose (also DMEM-LG herein) (Invitrogen,Carlsbad, Calif.). The DMEM—low glucose is preferably supplemented with15% (v/v) fetal bovine serum (e.g. defined fetal bovine serum, Hyclone,Logan Utah), antibiotics/antimycotics ((preferably 50-100Units/milliliter penicillin, 50-100 microgram/milliliter streptomycin,and 0-0.25 microgram/milliliter amphotericin B; Invitrogen, Carlsbad,Calif.)), and 0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis Mo.). Asused in the Examples below, Growth Medium refers to DMEM—low glucosewith 15% fetal bovine serum and antibiotics/antimycotics (whenpenicillin/streptomycin are included, it is preferably at 50 U/ml and 50microgram/ml respectively; when penicillin/streptomycin/amphotericin Bare use, it is preferably at 100 U/ml, 100 microgram/ml and 0.25microgram/ml, respectively). In some cases different growth media areused, or different supplementations are provided, and these are normallyindicated in the text as supplementations to Growth Medium.

The following abbreviations may appear in the examples and elsewhere inthe specification and claims: ANG2 (or Ang2) for angiopoietin 2; APC forantigen-presenting cells; BDNF for brain-derived neurotrophic factor;bFGF for basic fibroblast growth factor; bid (BID) for “bis in die”(twice per day); CK18 for cytokeratin 18; CNS for central nervoussystem; CXC ligand 3 for chemokine receptor ligand 3; DMEM forDulbecco's Minimal Essential Medium; DMEM:lg (or DMEM:Lg, DMEM:LG) forDMEM with low glucose; EDTA for ethylene diamine tetraacetic acid; EGF(or E) for epidermal growth factor; FACS for fluorescent activated cellsorting; FBS for fetal bovine serum; FGF (or F) for fibroblast growthfactor; GCP-2 for granulocyte chemotactic protein-2; GFAP for glialfibrillary acidic protein; HB-EGF for heparin-binding epidermal growthfactor; HCAEC for Human coronary artery endothelial cells; HGF forhepatocyte growth factor; hMSC for Human mesenchymal stem cells;HNF-1alpha for hepatocyte-specific transcription factor; HUVEC for Humanumbilical vein endothelial cells; 1309 for a chemokine and the ligandfor the CCR8 receptor; IGF-1 for insulin-like growth factor 1; IL-6 forinterleukin-6; IL-8 for interleukin 8; K19 for keratin 19; K8 forkeratin 8; KGF for keratinocyte growth factor; LIF for leukemiainhibitory factor; MBP for myelin basic protein; MCP-1 for monocytechemotactic protein 1; MDC for macrophage-derived chemokine; MIP1alphafor macrophage inflammatory protein 1 alpha; MIP1beta for macrophageinflammatory protein 1beta; MMP for matrix metalloprotease (MMP); MSCfor mesenchymal stem cells; NHDF for Normal Human Dermal Fibroblasts;NPE for Neural Progenitor Expansion media; 04 for oligodendrocyte orglial differentiation marker O4; PBMC for Peripheral blood mononuclearcell; PBS for phosphate buffered saline; PDGFbb for platelet derivedgrowth factor; PO for “per os” (by mouth); PNS for peripheral nervoussystem; Rantes (or RANTES) for regulated on activation, normal T cellexpressed and secreted; rhGDF-5 for recombinant human growth anddifferentiation factor 5; SC for subcutaneously; SDF-1alpha forstromal-derived factor 1 alpha; SHH for sonic hedgehog; SOP for standardoperating procedure; TARC for thymus and activation-regulated chemokine;TCP for Tissue culture plastic; TCPS for tissue culture polystyrene;TGFbeta2 for transforming growth factor beta2; TGF beta-3 fortransforming growth factor beta-3; TIMP1 for tissue inhibitor of matrixmetalloproteinase 1; TPO for thrombopoietin; TUJ1 for BIII Tubulin; VEGFfor vascular endothelial growth factor; vWF for von Willebrand factor;and alphaFP for alpha-fetoprotein.

The present invention is further illustrated, but not limited by, thefollowing examples.

Example 1 Derivation of Cells from Postpartum Tissue

This example describes the preparation of postpartum-derived cells fromplacental and umbilical cord tissues. Postpartum umbilical cords andplacentae were obtained upon birth of either a full term or pre-termpregnancy. Cells were harvested from five separate donors of umbilicusand placental tissue. Different methods of cell isolation were testedfor their ability to yield cells with: 1) the potential to differentiateinto cells with different phenotypes, a characteristic common to stemcells, or 2) the potential to provide trophic factors useful for othercells and tissues.

Methods & Materials Umbilical Cell Isolation:

Umbilical cords were obtained from National Disease Research Interchange(NDR1, Philadelphia, Pa.). The tissues were obtained following normaldeliveries. The cell isolation protocol was performed aseptically in alaminar flow hood. To remove blood and debris, the cord was washed inphosphate buffered saline (PBS; Invitrogen, Carlsbad, Calif.) in thepresence of antimycotic and antibiotic (100 units/milliliter penicillin,100 micrograms/milliliter streptomycin, 0.25 micrograms/milliliteramphotericin B). The tissues were then mechanically dissociated in 150cm² tissue culture plates in the presence of 50 milliliters of medium(DMEM—Low glucose or DMEM—High glucose; Invitrogen), until the tissuewas minced into a fine pulp. The chopped tissues were transferred to 50milliliter conical tubes (approximately 5 grams of tissue per tube). Thetissue was then digested in either DMEM—Low glucose medium or DMEM—Highglucose medium, each containing antimycotic and antibiotic as describedabove. In some experiments, an enzyme mixture of collagenase and dispasewas used (“C:D;” collagenase (Sigma, St Louis, Mo.), 500Units/milliliter; and dispase (Invitrogen), 50 Units/milliliter inDMEM:—Low glucose medium). In other experiments a mixture ofcollagenase, dispase and hyaluronidase (“C:D:H”) was used (collagenase,500 Units/milliliter; dispase, 50 Units/milliliter; and hyaluronidase(Sigma), 5 Units/milliliter, in DMEM:—Low glucose). The conical tubescontaining the tissue, medium and digestion enzymes were incubated at37° C. in an orbital shaker (Environ, Brooklyn, N.Y.) at 225 rpm for 2hrs.

After digestion, the tissues were centrifuged at 150×g for 5 minutes,and the supernatant was aspirated. The pellet was resuspended in 20milliliters of Growth Medium (DMEM: Low glucose (Invitrogen), 15 percent(v/v) fetal bovine serum (FBS; defined bovine serum; Lot#AND18475;Hyclone, Logan, Utah), 0.001% (v/v) 2-mercaptoethanol (Sigma), 1milliliter per 100 milliliters of antibiotic/antimycotic as describedabove. The cell suspension was filtered through a 70-micrometer nyloncell strainer (BD Biosciences). An additional 5 milliliters rinsecomprising Growth Medium was passed through the strainer. The cellsuspension was then passed through a 40-micrometer nylon cell strainer(BD Biosciences) and chased with a rinse of an additional 5 millilitersof Growth Medium.

The filtrate was resuspended in Growth Medium (total volume 50milliliters) and centrifuged at 150×g for 5 minutes. The supernatant wasaspirated and the cells were resuspended in 50 milliliters of freshGrowth Medium. This process was repeated twice more.

Upon the final centrifugation supernatant was aspirated and the cellpellet was resuspended in 5 milliliters of fresh Growth Medium. Thenumber of viable cells was determined using Trypan Blue staining. Cellswere then cultured under standard conditions.

The cells isolated from umbilical cords were seeded at 5,000 cells/cm²onto gelatin-coated T-75 cm² flasks (Corning Inc., Corning, N.Y.) inGrowth Medium with antibiotics/antimycotics as described above. After 2days (in various experiments, cells were incubated from 2-4 days), spentmedium was aspirated from the flasks. Cells were washed with PBS threetimes to remove debris and blood-derived cells. Cells were thenreplenished with Growth Medium and allowed to grow to confluence (about10 days from passage 0) to passage 1. On subsequent passages (frompassage 1 to 2 and so on), cells reached sub-confluence (75-85 percentconfluence) in 4-5 days. For these subsequent passages, cells wereseeded at 5000 cells/cm². Cells were grown in a humidified incubatorwith 5 percent carbon dioxide and atmospheric oxygen, at 37° C.

Placental Cell Isolation:

Placental tissue was obtained from NDR1 (Philadelphia, Pa.). The tissueswere from a pregnancy and were obtained at the time of a normal surgicaldelivery. Placental cells were isolated as described for umbilical cellisolation.

The following example applies to the isolation of separate populationsof maternal-derived and neonatal-derived cells from placental tissue.

The cell isolation protocol was performed aseptically in a laminar flowhood. The placental tissue was washed in phosphate buffered saline (PBS;Invitrogen, Carlsbad, Calif.) in the presence of antimycotic andantibiotic (as described above) to remove blood and debris. Theplacental tissue was then dissected into three sections: top-line(neonatal side or aspect), mid-line (mixed cell isolation neonatal andmaternal) and bottom line (maternal side or aspect).

The separated sections were individually washed several times in PBSwith antibiotic/antimycotic to further remove blood and debris. Eachsection was then mechanically dissociated in 150 cm² tissue cultureplates in the presence of 50 milliliters of DMEM/Low glucose, to a finepulp. The pulp was transferred to 50 milliliter conical tubes. Each tubecontained approximately 5 grams of tissue. The tissue was digested ineither DMEM—Low glucose or DMEM—High glucose medium containingantimycotic and antibiotic (100 U/milliliter penicillin, 100micrograms/milliliter streptomycin, 0.25 micrograms/milliliteramphotericin B) and digestion enzymes. In some experiments an enzymemixture of collagenase and dispase (“C:D”) was used containingcollagenase (Sigma, St Louis, Mo.) at 500 Units/milliliter and dispase(Invitrogen) at 50 Units/milliliter in DMEM—Low glucose medium. In otherexperiments a mixture of collagenase, dispase and hyaluronidase (C:D:H)was used (collagenase, 500 Units/milliliter; dispase, 50Units/milliliter; and hyaluronidase (Sigma), 5 Units/milliliter inDMEM—Low glucose). The conical tubes containing the tissue, medium, anddigestion enzymes were incubated for 2 h at 37° C. in an orbital shaker(Environ, Brooklyn, N.Y.) at 225 rpm.

After digestion, the tissues were centrifuged at 150×g for 5 minutes,the resultant supernatant was aspirated off. The pellet was resuspendedin 20 milliliters of Growth Medium withpenicillin/streptomycin/amphotericin B. The cell suspension was filteredthrough a 70 micometer nylon cell strainer (BD Biosciences), chased by arinse with an additional 5 milliliters of Growth Medium. The total cellsuspension was passed through a 40 micometer nylon cell strainer (BDBiosciences) followed with an additional 5 milliliters of Growth Mediumas a rinse.

The filtrate was resuspended in Growth Medium (total volume 50milliliters) and centrifuged at 150×g for 5 minutes. The supernatant wasaspirated and the cell pellet was resuspended in 50 milliliters of freshGrowth Medium. This process was repeated twice more. After the finalcentrifugation, supernatant was aspirated and the cell pellet wasresuspended in 5 milliliters of fresh Growth Medium. A cell count wasdetermined using the Trypan Blue Exclusion test. Cells were thencultured at standard conditions.

LIBERASE Cell Isolation:

Cells were isolated from umbilicus tissues in DMEM—Low glucose mediumwith LIBERASE (Boehringer Mannheim Corp., Indianapolis, Ind.) (2.5milligrams per milliliter, Blendzyme 3; Roche Applied Sciences,Indianapolis, Ind.) and hyaluronidase (5 Units/milliliter, Sigma).Digestion of the tissue and isolation of the cells was as described forother protease digestions above, using the LIBERASE/hyaluronidasemixture in place of the C:D or C:D:H enzyme mixture. Tissue digestionwith LIBERASE resulted in the isolation of cell populations frompostpartum tissues that expanded readily.

Cell Isolation Using Other Enzyme Combinations:

Procedures were compared for isolating cells from the umbilical cordusing differing enzyme combinations. Enzymes compared for digestionincluded: i) collagenase; ii) dispase; iii) hyaluronidase; iv)collagenase: dispase mixture (C;D); v) collagenase: hyaluronidasemixture (C:H); vi) dispase: hyaluronidase mixture (D:H); and vii)collagenase: dispase: hyaluronidase mixture (C:D:H). Differences in cellisolation utilizing these different enzyme digestion conditions wereobserved (Table 1-1).

Isolation of Cells from Residual Blood in the Cords:

Other attempts were made to isolate pools of cells from umbilical cordby different approaches. In one instance umbilical cord was sliced andwashed with Growth Medium to dislodge the blood clots and gelatinousmaterial. The mixture of blood, gelatinous material and Growth Mediumwas collected and centrifuged at 150×g. The pellet was resuspended andseeded onto gelatin-coated flasks in Growth Medium. From theseexperiments a cell population was isolated that readily expanded.

Isolation of Cells from Cord Blood:

Cells have also been isolated from cord blood samples attained fromNDR1. The isolation protocol used here was that of International PatentApplication US0229971 by Ho et al (Ho, T. W. et al., WO2003025149 A2).Samples (50 milliliter and 10.5 milliliters, respectively) of umbilicalcord blood (NDR1, Philadelphia Pa.) were mixed with lysis buffer(filter-sterilized 155 mM ammonium chloride, 10 millimolar potassiumbicarbonate, 0.1 millimolar EDTA buffered to pH 7.2 (all components fromSigma, St. Louis, Mo.)). Cells were lysed at a ratio of 1:20 cord bloodto lysis buffer. The resulting cell suspension was vortexed for 5seconds, and incubated for 2 minutes at ambient temperature. The lysatewas centrifuged (10 minutes at 200×g). The cell pellet was resuspendedin complete minimal essential medium (Gibco, Carlsbad Calif.) containing10 percent fetal bovine serum (Hyclone, Logan Utah), 4 millimolarglutamine (Mediatech Hemdon, Va.), 100 Units penicillin per 100milliliters and 100 micrograms streptomycin per 100 milliliters (Gibco,Carlsbad, Calif.). The resuspended cells were centrifuged (10 minutes at200×g), the supernatant was aspirated, and the cell pellet was washed incomplete medium. Cells were seeded directly into either T75 flasks(Corning, N.Y.), T75 laminin-coated flasks, or T175 fibronectin-coatedflasks (both Becton Dickinson, Bedford, Mass.).

Isolation of Cells Using Different Enzyme Combinations and GrowthConditions:

To determine whether cell populations could be isolated under differentconditions and expanded under a variety of conditions immediately afterisolation, cells were digested in Growth Medium with or without 0.001percent (v/v) 2-mercaptoethanol (Sigma, St. Louis, Mo.), using theenzyme combination of C:D:H, according to the procedures provided above.Placental-derived cells so isolated were seeded under a variety ofconditions. All cells were grown in the presence ofpenicillin/streptomycin. (Table 1-2).

Isolation of Cells Using Different Enzyme Combinations and GrowthConditions:

In all conditions cells attached and expanded well between passage 0 and1 (Table 1-2). Cells in conditions 5-8 and 13-16 were demonstrated toproliferate well up to 4 passages after seeding at which point they werecryopreserved and banked.

Results Cell Isolation Using Different Enzyme Combinations:

The combination of C:D:H, provided the best cell yield followingisolation, and generated cells, which expanded for many more generationsin culture than the other conditions (Table 1-1). An expandable cellpopulation was not attained using collagenase or hyaluronidase alone. Noattempt was made to determine if this result is specific to the collagenthat was tested.

Isolation of Cells Using Different Enzyme Combinations and GrowthConditions:

Cells attached and expanded well between passage 0 and 1 under allconditions tested for enzyme digestion and growth (Table 1-2). Cells inexperimental conditions 5-8 and 13-16 proliferated well up to 4 passagesafter seeding, at which point they were cryopreserved. All cells werebanked for further investigation.

Isolation of Cells from Residual Blood in the Cords:

Nucleated cells attached and grew rapidly. These cells were analyzed byflow cytometry and were similar to cells obtained by enzyme digestion.

Isolation of Cells from Cord Blood:

The preparations contained red blood cells and platelets. No nucleatedcells attached and divided during the first 3 weeks. The medium waschanged 3 weeks after seeding and no cells were observed to attach andgrow.

Summary:

Populations of cells can be derived from umbilical cord and placentaltissue efficiently using the enzyme combination collagenase (a matrixmetalloprotease), dispase (a neutral protease) and hyaluronidase (amucolytic enzyme that breaks down hyaluronic acid). LIBERASE, which is aBlendzyme, may also be used. Specifically, Blendzyme 3, which iscollagenase (4 Wunsch units/g) and thermolysin (1714 casein Units/g) wasalso used together with hyaluronidase to isolate cells. These cellsexpanded readily over many passages when cultured in Growth Medium ongelatin-coated plastic.

Cells were also isolated from residual blood in the cords, but not cordblood. The presence of cells in blood clots washed from the tissue thatadhere and grow under the conditions used may be due to cells beingreleased during the dissection process.

Example 2 Growth Characteristics of Postpartum-Derived Cells

The cell expansion potential of postpartum-derived cells (PPDCs) wascompared to other populations of isolated stem cells. The process ofcell expansion to senescence is referred to as Hayflick's limit(Hayflick L. 1974a, 1974b). Postpartum-derived cells are highly suitedfor therapeutic use because they can be readily expanded to sufficientcell numbers.

Materials and Methods Gelatin-Coating Flasks:

Tissue culture plastic flasks were coated by adding 20 milliliters 2%(w/v) porcine gelatin (Type B: 225 Bloom; Sigma, St Louis, Mo.) to a T75flask (Corning, Corning, N.Y.) for 20 minutes at room temperature. Afterremoving the gelatin solution, 10 milliliters phosphate-buffered saline(PBS) (Invitrogen, Carlsbad, Calif.) was added and then aspirated.

Comparison of Expansion Potential of PPDCs with Other Cell Populations:

For comparison of growth expansion potential the following cellpopulations were utilized; i) Mesenchymal stem cells (MSC; Cambrex,Walkersville, Md.); ii) Adipose-derived cells (U.S. Pat. No. 6,555,374B1; U.S. Patent Application US20040058412); iii) Normal dermal skinfibroblasts (cc-2509 lot #9F0844; Cambrex, Walkersville, Md.); iv)Umbilicus-derived cells; and v) Placenta-derived cells. Cells wereinitially seeded at 5,000 cells/cm² on gelatin-coated T75 flasks inGrowth Medium with penicillin/streptomycin/amphotericin B. Forsubsequent passages, cell cultures were treated as follows. Aftertrypsinization, viable cells were counted after Trypan Blue stainingCell suspension (50 microliters) was combined with Trypan Blue (50milliliters, Sigma, St. Louis Mo.). Viable cell numbers were estimatedusing a hemocytometer.

Following counting, cells were seeded at 5,000 cells/cm² ontogelatin-coated T 75 flasks in 25 milliliters of fresh Growth Medium.Cells were grown under standard conditions at 37° C. The Growth Mediumwas changed twice per week. When cells reached about 85 percentconfluence they were passaged; this process was repeated until the cellsreached senescence.

At each passage, cells were trypsinized and counted. The viable cellyield, population doubling [ln (cell final/cell initial)/ln 2] anddoubling time (time in culture (h)/population doubling) were calculated.For the purposes of determining optimal cell expansion, the total cellyield per passage was determined by multiplying the total yield for theprevious passage by the expansion factor for each passage (i.e.,expansion factor=cell final/cell initial).

Expansion Potential of Cell Banks at Low Density:

The expansion potential of cells banked at passage 10 was also tested,using a different set of conditions. Normal dermal skin fibroblasts(cc-2509 lot #9F0844; Cambrex, Walkersville, Md.), umbilicus-derivedcells, and placenta-derived cells were tested. These cell populationshad been banked at passage 10 previously, having been cultured at 5,000cells/cm² and grown to confluence at each passage to that point. Theeffect of cell density on the cell populations following cell thaw atpassage 10 was determined Cells were thawed under standard conditionsand counted using Trypan Blue staining. Thawed cells were then seeded at1000 cells/cm² in DMEM:Low glucose Growth Medium withantibiotic/antimycotic as described above. Cells were grown understandard atmospheric conditions at 37° C. Growth Medium was changedtwice a week and cells were passaged as they reached about 85%confluence. Cells were subsequently passaged until senescence, i.e.,until they could not be expanded any further. Cells were trypsinized andcounted at each passage. The cell yield, population doubling (ln (cellfinal/cell initial)/ln 2) and doubling time (time in culture(h)/population doubling). The total cell yield per passage wasdetermined by multiplying total yield for the previous passage by theexpansion factor for each passage (i.e., expansion factor=cellfinal/cell initial).

Expansion of PPDCs at Low Density from Initial Cell Seeding:

The expansion potential of freshly isolated PPDCs under low cell seedingconditions was tested. PPDDs were prepared as described herein. Cellswere seeded at 1000 cells/cm² and passaged as described above untilsenescence. Cells were grown under standard atmospheric conditions at37° C. Growth Medium was changed twice per week. Cells were passaged asthey reached about 85% confluence. At each passage, cells weretrypsinized and counted by Trypan Blue staining. The cell yield,population doubling (in (cell final/cell initial)/ln 2) and doublingtime (time in culture (h)/population doubling) were calculated for eachpassage. The total cell yield per passage was determined by multiplyingthe total yield for the previous passage by the expansion factor foreach passage (i.e. expansion factor=cell final/cell initial). Cells weregrown on gelatin and non-gelatin coated flasks.

Expansion of Clonal Neonatal Placenta-Derived Cells:

Cloning was used in order to expand a population of neonatal cells fromplacental tissue. Following isolation of three differential cellpopulations from the placenta (as described herein), these cellpopulations were expanded under standard growth conditions and thenkaryotyped to reveal the identity of the isolated cell populations.Because the cells were isolated from a mother who delivered a boy, itwas straightforward to distinguish between the male and femalechromosomes by performing metaphase spreads. These experimentsdemonstrated that fetal-aspect cells were karyotype positive forneonatal phenotype, mid-layer cells were karyotype positive for bothneonatal and maternal phenotypes and maternal-aspect cells werekaryotype positive for maternal cells.

Expansion of Cells in Low Oxygen Culture Conditions:

It has been demonstrated that low oxygen cell culture conditions canimprove cell expansion in certain circumstances (US20040005704). Todetermine if cell expansion of PPDCs could be improved by altering cellculture conditions, cultures of umbilical-derived cells were grown inlow oxygen conditions. Cells were seeded at 5000 cells/cm² in GrowthMedium on gelatin coated flasks. Cells were initially cultured understandard atmospheric conditions through passage 5, at which point theywere transferred to low oxygen (5% O₂) culture conditions.

Other Growth Conditions:

In other protocols, cells were expanded on non-coated, collagen-coated,fibronectin-coated, laminin-coated and extracellular matrixprotein-coated plates. Cultures have been demonstrated to expand well onthese different matrices.

Results

Comparison of Expansion Potential of PPDCs with Other Stem Cell andNon-Stem Cell Populations:

Both umbilical-derived and placenta-derived cells expanded for greaterthan 40 passages generating cell yields of greater than 1E¹⁷ cells in 60days. In contrast, MSCs and fibroblasts senesced after less than 25 daysand less than 60 days, respectively. Although adipose-derived cellsexpanded for almost 60 days they generated total cell yields of 4.5E¹².Thus, when seeded at 5000 cells/cm² under the experimental conditionsutilized, postpartum-derived cells expanded much better than the othercell types grown under the same conditions (Table 2-1).

Expansion Potential of Cell Banks at Low Density:

Umbilicus-derived, placenta-derived and fibroblast cells expanded forgreater than 10 passages generating cell yields of greater than 1E¹¹cells in 60 days (Table 2-2). After 60 days under these conditions thefibroblasts became senescent whereas the umbilicus-derived andplacenta-derived cell populations senesced after 80 days, completinggreater than 50 and greater than 40 population doublings respectively.

Expansion of PPDCs at low density from initial cell seeding. PPDCs wereexpanded at low density (1,000 cells/cm²) on gelatin-coated and uncoatedplates or flasks. Growth potential of these cells under these conditionswas good. The cells expanded readily in a log phase growth. The rate ofcell expansion was similar to that observed when placenta-derived cellswere seeded at 5000 cells/cm² on gelatin-coated flasks in Growth Medium.No differences were observed in cell expansion potential betweenculturing on either uncoated flasks or gelatin-coated flasks. However,cells appeared phenotypically much smaller on gelatin-coated flasks andmore larger cell phenotypes were observed on uncoated flasks.

Expansion of Clonal Neonatal or Maternal Placenta-Derived Cells:

A clonal neonatal or maternal cell population can be expanded fromplacenta-derived cells isolated from the neonatal aspect or the maternalaspect, respectively, of the placenta. Cells are serially diluted andthen seeded onto gelatin-coated plates in Growth medium for expansion at1 cell/well in 96-well gelatin coated plates. From this initial cloning,expansive clones are identified, trypsinized, and reseeded in 12-wellgelatin-coated plates in Growth medium and then subsequently passagedinto T25 gelatin-coated flasks at 5,000 cells/cm² in Growth medium.Subcloning is performed to ensure that a clonal population of cells hasbeen identified. For subcloning experiments, cells are trypsinized andreseeded at 0.5 cells/well. The subclones that grow well are expanded ingelatin-coated T25 flasks at 5,000 cells cm²/flask. Cells are passagedat 5,000 cells cm²/T75 flask. The growth characteristics of a clone maybe plotted to demonstrate cell expansion. Karyotyping analysis canconfirm that the clone is either neonatal or maternal.

Expansion of Cells in Low Oxygen Culture Conditions:

Cells expanded well under the reduced oxygen conditions, however,culturing under low oxygen conditions did not appear to have asignificant effect on cell expansion of PPDCs under the conditions used.

Summary:

Cell expansion conditions comprising growing isolated postpartum-derivedcells at densities of about 5000 cells/cm², in Growth Medium ongelatin-coated or uncoated flasks, under standard atmospheric oxygen,are sufficient to generate large numbers of cells at passage 11.Furthermore, the data suggests that the cells can be readily expandedusing lower density culture conditions (e.g. 1000 cells/cm²).Postpartum-derived cell expansion in low oxygen conditions alsofacilitates cell expansion, although no incremental improvement in cellexpansion potential has yet been observed when utilizing theseconditions for growth. Presently, culturing postpartum-derived cellsunder standard atmospheric conditions is preferred for generating largepools of cells. However, when the culture conditions are altered,postpartum-derived cell expansion can likewise be altered. This strategymay be used to enhance the proliferative and differentiative capacity ofthese cell populations.

Under the conditions utilized, while the expansion potential of MSC andadipose-derived cells is limited, postpartum-derived cells expandreadily to large numbers.

Example 3 Evaluation of Growth Media for Placenta-Derived Cells

Several cell culture media were evaluated for their ability to supportthe growth of placenta-derived cells. The growth of placenta-derivedcells in normal (20%) and low (5%) oxygen was assessed after 3 daysusing the MTS calorimetric assay.

Methods & Materials

Placenta-derived cells at passage 8 (P8) were seeded at 1×10³ cells/wellin 96 well plates in Growth Medium with penicillin/streptomycin. After 8hours the medium was changed as described below and cells were incubatedin normal (atmospheric) or low (5%, v/v) oxygen at 37° C., 5% CO₂ for 48hours. MTS was added to the culture medium (CELLTITER 96 AQueous OneSolution Cell Proliferation Assay, Promega, Madison, Wis.) for 3 hoursand the absorbance measured at 490 nanometers (Molecular Devices,Sunnyvale Calif.).

Results

Standard curves for the MTS assay established a linear correlationbetween an increase in absorbance and an increase in cell number. Theabsorbance values obtained were converted into estimated cell numbersand the change (%) relative to the initial seeding was calculated.

The Effect of Serum:

The addition of serum to media at normal oxygen conditions resulted in areproducible dose-dependent increase in absorbance and thus the viablecell number. The addition of serum to complete MSCGM resulted in adose-dependent decrease in absorbance. In the media without added serum,cells only grew appreciably in CELLGRO-FREE, Ham's F10 and DMEM.

The Effect of Oxygen:

Reduced oxygen appeared to increase the growth rate of cells in GrowthMedium, Ham's F10, and MSCGM. In decreasing order of growth, the mediaresulting in the best growth of the cells were Growth Medium, greaterthan MSCGM, greater than Iscove's+10% FBS, equal to DMEM-H+10% FBS,equal to Ham's F12+10% FBS, equal to RPMI 1640+10% FBS.

Summary:

Placenta-derived cells may be grown in a variety of culture media innormal or low oxygen. Short-term growth of placenta-derived cells wasdetermined in twelve basal media with 0, 2 and 10% (v/v) serum in 5% oratmospheric oxygen. In general, placenta-derived cells did not grow aswell in serum-free conditions with the exception of Ham's F10 andCELLGRO-Free, which are also protein-free. Growth in these serum-freemedia was about 25-33% of the maximal growth observed with mediacontaining 15% serum.

Example 4 Growth of Postpartum-Derived Cells in Medium ContainingD-Valine

It has been reported that medium containing D-valine instead of thenormal L-valine isoform can be used to selectively inhibit the growth offibroblast-like cells in culture (Hongpaisan, 2000; Sordillo et al.,1988). It was not previously known whether postpartum-derived cellscould grow in medium containing D-valine.

Methods & Materials

Placenta-derived cells (P3), fibroblasts (P9) and umbilical-derivedcells (P5) were seeded at 5×10³ cells/cm² in gelatin-coated T75 flasks(Corning, Corning, N.Y.). After 24 hours the medium was removed and thecells were washed with phosphate buffered saline (PBS) (Gibco, Carlsbad,Calif.) to remove residual medium. The medium was replaced with aModified Growth Medium (DMEM with D-valine (special order Gibco), 15%(v/v) dialyzed fetal bovine serum (Hyclone, Logan, Utah), 0.001% (v/v)betamercaptoethanol (Sigma), penicillin/streptomycin (Gibco)).

Results

Placenta-derived, umbilical-derived, and fibroblast cells seeded in theD-valine-containing medium did not proliferate, unlike cells seeded inGrowth Medium containing dialyzed serum. Fibroblasts cells changedmorphologically, increasing in size and changing shape. All of the cellsdied and eventually detached from the flask surface after 4 weeks. Theseresults indicate that medium containing D-valine is not suitable forselectively growing postpartum-derived cells.

Example 5 Cryopreservation Media for Placenta-Derived Cells

Cryopreservation media for the cryopreservation of placenta-derivedcells were evaluated.

Methods & Materials

Placenta-derived cells grown in Growth Medium in a gelatin-coated T75flask were washed with PBS and trypsinized using 1 milliliterTrypsin/EDTA (Gibco). The trypsinization was stopped by adding 10milliliters Growth Medium. The cells were centrifuged at 150×g,supernatant removed, and the cell pellet was resuspended in 1 milliliterGrowth Medium. An aliquot of cell suspension, 60 microliters, wasremoved and added to 60 microliters trypan blue (Sigma). The viable cellnumber was estimated using a hemocytometer. The cell suspension wasdivided into four equal aliquots each containing 88×10⁴ cells each. Thecell suspension was centrifuged and resuspended in 1 milliliter of eachmedia below and transferred into Cryovials (Nalgene).

-   -   a. Growth Medium+10% (v/v) DMSO (Hybrimax, Sigma, St. Louis,        Mo.)    -   b. Cell Freezing medium w/DMSO, w/methyl cellulose, serum-free        (C6295, Sigma, St. Louis, Mo.)    -   c. Cell Freezing medium serum-free (C2639, Sigma, St. Louis,        Mo.)    -   d. Cell Freezing Medium w/glycerol (C6039, Sigma, St. Louis,        Mo.)

The cells were cooled at approximately-1° C./min overnight in a −80° C.freezer using a “Mr Frosty” freezing container according to themanufacturer's instructions (Nalgene, Rochester, N.Y.). Vials of cellswere transferred into liquid nitrogen for 2 days before thawing rapidlyin a 37° C. water bath. The cells were added to 10 milliliters GrowthMedium and centrifuged before the cell number and viability wasestimated. Cells were seeded onto gelatin-coated flasks at 5,000cells/cm² to determine whether the cells would attach and proliferate.

Results

The initial viability of the cells to be cryopreserved was assessed bytrypan blue staining to be 100%. The initial viability of the cells tobe cryopreserved was assessed by trypan blue staining to be 100%.

There was a commensurate reduction in cell number with viability forC6295 due to cells lysis. The viable cells cryopreserved in all foursolutions attached, divided, and produced a confluent monolayer within 3days. There was no discernable difference in estimated growth rate.

Summary:

The cryopreservation of cells is one procedure available for preparationof a cell bank or a cell product. Four cryopreservation mixtures werecompared for their ability to protect human placenta-derived cells fromfreezing damage. Dulbecco's modified Eagle's medium (DMEM) and 10% (v/v)dimethylsulfoxide (DMSO) is the preferred medium of those compared forcryopreservation of placenta-derived cells.

Example 6 Karyotype Analysis of Postpartum-Derived Cells

Cell lines used in cell therapy are preferably homogeneous and free fromany contaminating cell type. Cells used in cell therapy should have anormal chromosome number (46) and structure. To identify placenta- andumbilicus-derived cell lines that are homogeneous and free from cells ofnon-postpartum tissue origin, karyotypes of cell samples were analyzed.

Methods & Materials

PPDCs from postpartum tissue of a male neonate were cultured in GrowthMedium containing penicillin/streptomycin. Postpartum tissue from a maleneonate (X,Y) was selected to allow distinction between neonatal-derivedcells and maternal derived cells (X,X). Cells were seeded at 5,000 cellsper square centimeter in Growth Medium in a T25 flask (Corning, Corning,N.Y.) and expanded to 80% confluence. A T25 flask containing cells wasfilled to the neck with Growth Medium. Samples were delivered to aclinical cytogenetics laboratory by courier (estimated lab to labtransport time is one hour). Cells were analyzed during metaphase whenthe chromosomes are best visualized. Of twenty cells in metaphasecounted, five were analyzed for normal homogeneous karyotype number(two). A cell sample was characterized as homogeneous if two karyotypeswere observed. A cell sample was characterized as heterogeneous if morethan two karyotypes were observed. Additional metaphase cells werecounted and analyzed when a heterogeneous karyotype number (four) wasidentified.

Results

All cell samples sent for chromosome analysis were interpreted asexhibiting a normal appearance. Three of the sixteen cell lines analyzedexhibited a heterogeneous phenotype (XX and XY) indicating the presenceof cells derived from both neonatal and maternal origins (Table 6-1).Cells derived from tissue Placenta-N were isolated from the neonatalaspect of placenta. At passage zero, this cell line appeared homogeneousXY. However, at passage nine, the cell line was heterogeneous (XX/XY),indicating a previously undetected presence of cells of maternal origin.

Summary:

Chromosome analysis identified placenta- and umbilicus-derived cellswhose karyotypes appeared normal as interpreted by a clinicalcytogenetic laboratory. Karyotype analysis also identified cell linesfree from maternal cells, as determined by homogeneous karyotype.

Example 7 Evaluation of Human Postpartum-Derived Cell Surface Markers byFlow Cytometry

Characterization of cell surface proteins or “markers” by flow cytometrycan be used to determine a cell line's identity. The consistency ofexpression can be determined from multiple donors, and in cells exposedto different processing and culturing conditions. Postpartum-derivedcell (PPDC) lines isolated from the placenta and umbilicus werecharacterized (by flow cytometry), providing a profile for theidentification of these cell lines.

Methods & Materials Media and Culture Vessels:

Cells were cultured in Growth Medium (Gibco Carlsbad, Calif.) withpenicillin/streptomycin. Cells were cultured in plasma-treated T75,T150, and T225 tissue culture flasks (Corning, Corning, N.Y.) untilconfluent. The growth surfaces of the flasks were coated with gelatin byincubating 2% (w/v) gelatin (Sigma, St. Louis, Mo.) for 20 minutes atroom temperature.

Antibody Staining and Flow Cytometry Analysis:

Adherent cells in flasks were washed in PBS and detached withTrypsin/EDTA. Cells were harvested, centrifuged, and resuspended in 3%(v/v) FBS in PBS at a cell concentration of 1×10⁷ per milliliter. Inaccordance to the manufacture's specifications, antibody to the cellsurface marker of interest (see below) was added to one hundredmicroliters of cell suspension and the mixture was incubated in the darkfor 30 minutes at 4° C. After incubation, cells were washed with PBS andcentrifuged to remove unbound antibody. Cells were resuspended in 500microliter PBS and analyzed by flow cytometry. Flow cytometry analysiswas performed with a FACScalibur instrument (Becton Dickinson, San Jose,Calif.). Table 7 lists the antibodies to cell surface markers that wereused.

Placenta and Umbilicus Comparison:

Placenta-derived cells were compared to umbilicus-derive cells atpassage 8.

Passage to Passage Comparison:

Placenta- and umbilicus-derived cells were analyzed at passages 8, 15,and 20.

Donor to Donor Comparison:

To compare differences among donors, placenta-derived cells fromdifferent donors were compared to each other, and umbilicus-derivedcells from different donors were compared to each other.

Surface Coating Comparison:

Placenta-derived cells cultured on gelatin-coated flasks was compared toplacenta-derived cells cultured on uncoated flasks. Umbilicus-derivedcells cultured on gelatin-coated flasks was compared toumbilicus-derived cells cultured on uncoated flasks.

Digestion Enzyme Comparison:

Four treatments used for isolation and preparation of cells werecompared. Cells isolated from placenta by treatment with 1) collagenase;2) collagenase/dispase; 3) collagenase/hyaluronidase; and 4)collagenase/hyaluronidase/dispase were compared.

Placental Layer Comparison:

Cells derived from the maternal aspect of placental tissue were comparedto cells derived from the villous region of placental tissue and cellsderived from the neonatal fetal aspect of placenta.

Results

Placenta Vs. Umbilicus Comparison:

Placenta- and umbilicus-derived cells analyzed by flow cytometry showedpositive expression of CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha andHLA-A, B, C, indicated by the increased values of fluorescence relativeto the IgG control. These cells were negative for detectable expressionof CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, indicated byfluorescence values comparable to the IgG control. Variations influorescence values of positive curves were accounted for. The mean(i.e. CD13) and range (i.e. CD90) of the positive curves showed somevariation, but the curves appeared normal, confirming a homogenouspopulation. Both curves individually exhibited values greater than theIgG control.

Passage to Passage Comparison—Placenta-Derived Cells:

Placenta-derived cells at passages 8, 15, and 20 analyzed by flowcytometry all were positive for expression of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, as reflected in the increased value offluorescence relative to the IgG control. The cells were negative forexpression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ havingfluorescence values consistent with the IgG control.

Passage to Passage Comparison—Umbilicus-Derived Cells:

Umbilicus-derived cells at passage 8, 15, and 20 analyzed by flowcytometry all expressed CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha andHLA-A, B, C, indicated by increased fluorescence relative to the IgGcontrol. These cells were negative for CD31, CD34, CD45, CD117, CD141,and HLA-DR, DP, DQ, indicated by fluorescence values consistent with theIgG control.

Donor to Donor Comparison—Placenta-Derived Cells:

Placenta-derived cells isolated from separate donors analyzed by flowcytometry each expressed CD 10, CD 13, CD44, CD73, CD 90, PDGFr-alphaand HLA-A, B, C, with increased values of fluorescence relative to theIgG control. The cells were negative for expression of CD31, CD34, CD45,CD117, CD141, and HLA-DR, DP, DQ as indicated by fluorescence valueconsistent with the IgG control.

Donor to Donor Comparison—Umbilicus Derived Cells:

Umbilicus-derived cells isolated from separate donors analyzed by flowcytometry each showed positive expression of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, reflected in the increased values offluorescence relative to the IgG control. These cells were negative forexpression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ withfluorescence values consistent with the IgG control.

The Effect of Surface Coating with Gelatin on Placenta-Derived Cells:

Placenta-derived cells expanded on either gelatin-coated or uncoatedflasks analyzed by flow cytometry all expressed of CD10, CD13, CD44,CD73, CD 90, PDGFr-alpha and HLA-A, B, C, reflected in the increasedvalues of fluorescence relative to the IgG control. These cells werenegative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR,DP, DQ indicated by fluorescence values consistent with the IgG control.

The Effect of Surface Coating with Gelatin on Umbilicus-Derived Cells:

Umbilicus-derived cells expanded on gelatin and uncoated flasks analyzedby flow cytometry all were positive for expression of CD10, CD13, CD44,CD73, CD90, PDGFr-alpha and HLA-A, B, C, with increased values offluorescence relative to the IgG control. These cells were negative forexpression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, withfluorescence values consistent with the IgG control.

Effect of Enzyme Digestion Procedure Used for Preparation of the Cellson the Cell Surface Marker Profile:

Placenta-derived cells isolated using various digestion enzymes analyzedby flow cytometry all expressed CD 10, CD 13, CD44, CD73, CD 90,PDGFr-alpha and HLA-A, B, C, as indicated by the increased values offluorescence relative to the IgG control. These cells were negative forexpression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ asindicated by fluorescence values consistent with the IgG control.

Placental Layer Comparison:

Cells isolated from the maternal, villous, and neonatal layers of theplacenta, respectively, analyzed by flow cytometry showed positiveexpression of CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B,C, as indicated by the increased value of fluorescence relative to theIgG control. These cells were negative for expression of CD31, CD34,CD45, CD117, CD141, and HLA-DR, DP, DQ as indicated by fluorescencevalues consistent with the IgG control.

Summary:

Analysis of placenta- and umbilicus-derived cells by flow cytometry hasestablished of an identity of these cell lines. Placenta- andumbilicus-derived cells are positive for CD10, CD13, CD44, CD73, CD90,PDGFr-alpha, HLA-A, B, C and negative for CD31, CD34, CD45, CD117, CD141and HLA-DR, DP, DQ. This identity was consistent between variations invariables including the donor, passage, culture vessel surface coating,digestion enzymes, and placental layer. Some variation in individualfluorescence value histogram curve means and ranges was observed, butall positive curves under all conditions tested were normal andexpressed fluorescence values greater than the IgG control, thusconfirming that the cells comprise a homogenous population that haspositive expression of the markers.

Example 8 Immunohistochemical Characterization of Postpartum TissuePhenotypes

The phenotypes of cells found within human postpartum tissues, namelyumbilical cord and placenta, was analyzed by immunohistochemistry.

Methods & Materials Tissue Preparation:

Human umbilical cord and placenta tissue was harvested and immersionfixed in 4% (w/v) paraformaldehyde overnight at 4° C.Immunohistochemistry was performed using antibodies directed against thefollowing epitopes: vimentin (1:500; Sigma, St. Louis, Mo.), desmin(1:150, raised against rabbit; Sigma; or 1:300, raised against mouse;Chemicon, Temecula, Calif.), alpha-smooth muscle actin (SMA; 1:400;Sigma), cytokeratin 18 (CK18; 1:400; Sigma), von Willebrand Factor (vWF;1:200; Sigma), and CD34 (human CD34 Class III; 1:100; DAKOCytomation,Carpinteria, Calif.). In addition, the following markers were tested:anti-human GROalpha--PE (1:100; Becton Dickinson, Franklin Lakes, N.J.),anti-human GCP-2 (1:100; Santa Cruz Biotech, Santa Cruz, Calif.),anti-human oxidized LDL receptor 1 (ox-LDL R1; 1:100; Santa CruzBiotech), and anti-human NOGO-A (1:100; Santa Cruz Biotech). Fixedspecimens were trimmed with a scalpel and placed within OCT embeddingcompound (Tissue-Tek OCT; Sakura, Torrance, Calif.) on a dry ice bathcontaining ethanol. Frozen blocks were then sectioned (10 μm thick)using a standard cryostat (Leica Microsystems) and mounted onto glassslides for staining.

Immunohistochemistry:

Immunohistochemistry was performed similar to previous studies (e.g.,Messina, et al., 2003, Exper. Neurol. 184: 816-829). Tissue sectionswere washed with phosphate-buffered saline (PBS) and exposed to aprotein blocking solution containing PBS, 4% (v/v) goat serum (Chemicon,Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 1hour to access intracellular antigens. In instances where the epitope ofinterest would be located on the cell surface (CD34, ox-LDL R1), Tritonwas omitted in all steps of the procedure in order to prevent epitopeloss. Furthermore, in instances where the primary antibody was raisedagainst goat (GCP-2, ox-LDL R1, NOGO-A), 3% (v/v) donkey serum was usedin place of goat serum throughout the procedure. Primary antibodies,diluted in blocking solution, were then applied to the sections for aperiod of 4 hours at room temperature. Primary antibody solutions wereremoved, and cultures washed with PBS prior to application of secondaryantibody solutions (1 hour at room temperature) containing block alongwith goat anti-mouse IgG—Texas Red (1:250; Molecular Probes, Eugene,Oreg.) and/or goat anti-rabbit IgG—Alexa 488 (1:250; Molecular Probes)or donkey anti-goat IgG—FITC (1:150; Santa Cruz Biotech). Cultures werewashed, and 10 micromolar DAPI (Molecular Probes) was applied for 10minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). Positive staining was representedby fluorescence signal above control staining. Representative imageswere captured using a digital color video camera and ImagePro software(Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, eachimage was taken using only one emission filter at a time. Layeredmontages were then prepared using Adobe Photoshop software (Adobe, SanJose, Calif.).

Results Umbilical Cord Characterization:

Vimentin, desmin, SMA, CK18, vWF, and CD34 markers were expressed in asubset of the cells found within umbilical cord. In particular, vWF andCD34 expression were restricted to blood vessels contained within thecord. CD34+ cells were on the innermost layer (lumen side). Vimentinexpression was found throughout the matrix and blood vessels of thecord. SMA was limited to the matrix and outer walls of the artery &vein, but not contained with the vessels themselves. CK18 and desminwere observed within the vessels only, desmin being restricted to themiddle and outer layers.

Placenta Characterization:

Vimentin, desmin, SMA, CK18, vWF, and CD34 were all observed within theplacenta and regionally specific.

GROalpha, GCP-2, ox-LDL R1, and NOGO-A Tissue Expression:

None of these markers were observed within umbilical cord or placentaltissue.

Summary:

Vimentin, desmin, alpha-smooth muscle actin, cytokeratin 18, vonWillebrand Factor, and CD34 are expressed in cells within humanumbilical cord and placenta.

Example 9 Analysis of Postpartum Tissue-Derived Cells UsingOligonucleotide Arrays

Affymetrix GENECHIP arrays were used to compare gene expression profilesof umbilicus- and placenta-derived cells with fibroblasts, humanmesenchymal stem cells, and another cell line derived from human bonemarrow. This analysis provided a characterization of thepostpartum-derived cells and identified unique molecular markers forthese cells.

Methods & Materials Isolation and Culture of Cells:

Human umbilical cords and placenta were obtained from National DiseaseResearch Interchange (NDRI, Philadelphia, Pa.) from normal full termdeliveries with patient consent. The tissues were received and cellswere isolated as described in Example 1. Cells were cultured in GrowthMedium (using DMEM-LG) on gelatin-coated tissue culture plastic flasks.The cultures were incubated at 37° C. with 5% CO₂.

Human dermal fibroblasts were purchased from Cambrex Incorporated(Walkersville, Md.; Lot number 9F0844) and ATCC CRL-1501 (CCD39SK). Bothlines were cultured in DMEM/F12 medium (Invitrogen, Carlsbad, Calif.)with 10% (v/v) fetal bovine serum (Hyclone) and penicillin/streptomycin(Invitrogen). The cells were grown on standard tissue-treated plastic.

Human mesenchymal stem cells (hMSC) were purchased from CambrexIncorporated (Walkersville, Md.; Lot numbers 2F1655, 2F1656 and 2F1657)and cultured according to the manufacturer's specifications in MSCGMMedia (Cambrex). The cells were grown on standard tissue culturedplastic at 37° C. with 5% CO₂.

Human ileac crest bone marrow was received from the NDRI with patientconsent. The marrow was processed according to the method outlined byHo, et al. (WO03/025149). The marrow was mixed with lysis buffer (155 mMNH 4Cl, 10 mM KHCO₃, and 0.1 mM EDTA, pH 7.2) at a ratio of 1 part bonemarrow to 20 parts lysis buffer. The cell suspension was vortexed,incubated for 2 minutes at ambient temperature, and centrifuged for 10minutes at 500×g. The supernatant was discarded and the cell pellet wasresuspended in Minimal Essential Medium-alpha (Invitrogen) supplementedwith 10% (v/v) fetal bovine serum and 4 mM glutamine. The cells werecentrifuged again and the cell pellet was resuspended in fresh medium.The viable mononuclear cells were counted using trypan-blue exclusion(Sigma, St. Louis, Mo.). The mononuclear cells were seeded intissue-cultured plastic flasks at 5×10⁴ cells/cm². The cells wereincubated at 37° C. with 5% CO₂ at either standard atmospheric O₂ or at5% O₂. Cells were cultured for 5 days without a media change. Media andnon-adherent cells were removed after 5 days of culture. The adherentcells were maintained in culture.

Isolation of mRNA and GENECHIP Analysis:

Actively growing cultures of cells were removed from the flasks with acell scraper in cold PBS. The cells were centrifuged for 5 minutes at300×g. The supernatant was removed and the cells were resuspended infresh PBS and centrifuged again. The supernatant was removed and thecell pellet was immediately frozen and stored at −80° C. Cellular mRNAwas extracted and transcribed into cDNA, which was then transcribed intocRNA and biotin-labeled. The biotin-labeled cRNA was hybridized withHG-U133A GENECHIP oligonucleotide array (Affymetrix, Santa ClaraCalif.). The hybridization and data collection was performed accordingto the manufacturer's specifications. Analyses were performed using“Significance Analysis of Microarrays” (SAM) version 1.21 computersoftware (Stanford University, www-stat.stanford.edu/tibs/SAM; Tusher,V. G. et al., 2001, Proc. Natl. Acad. Sci. USA 98: 5116-5121).

Results

Fourteen different populations of cells were analyzed. The cells alongwith passage information, culture substrate, and culture media arelisted in Table 9-1.

The data were evaluated by a Principle Component Analysis, analyzing the290 genes that were differentially expressed in the cells. This analysisallows for a relative comparison for the similarities between thepopulations. Table 9-2 shows the Euclidean distances that werecalculated for the comparison of the cell pairs. The Euclidean distanceswere based on the comparison of the cells based on the 290 genes thatwere differentially expressed among the cell types. The Euclideandistance is inversely proportional to similarity between the expressionof the 290 genes (i.e., the greater the distance, the less similarityexists).

Tables 9-3, 9-4, and 9-5 show the expression of genes increased inplacenta-derived cells (Table 9-3), increased in umbilicus-derived cells(Table 9-4), and reduced in umbilicus- and placenta-derived cells (Table9-5). The column entitled “Probe Set ID” refers to the manufacturer'sidentification code for the sets of several oligonucleotide probeslocated on a particular site on the chip, which hybridize to the namedgene (column “Gene Name”), comprising a sequence that can be foundwithin the NCBI (GenBank) database at the specified accession number(column “NCBI Accession Number”).

Tables 9-6, 9-7, and 9-8 show the expression of genes increased in humanfibroblasts (Table 9-6), ICBM cells (Table 9-7), and MSCs (Table 9-8).

Summary:

The present examination was performed to provide a molecularcharacterization of the postpartum cells derived from umbilical cord andplacenta. This analysis included cells derived from three differentumbilical cords and three different placentas. The examination alsoincluded two different lines of dermal fibroblasts, three lines ofmesenchymal stem cells, and three lines of ileac crest bone marrowcells. The mRNA that was expressed by these cells was analyzed using anoligonucleotide array that contained probes for 22,000 genes. Resultsshowed that 290 genes are differentially expressed in these fivedifferent cell types. These genes include ten genes that arespecifically increased in the placenta-derived cells and seven genesspecifically increased in the umbilical cord-derived cells. Fifty-fourgenes were found to have specifically lower expression levels inplacenta and umbilical cord, as compared with the other cell types. Theexpression of selected genes has been confirmed by PCR (see the examplethat follows). These results demonstrate that the postpartum-derivedcells have a distinct gene expression profile, for example, as comparedto bone marrow-derived cells and fibroblasts.

Example 10 Cell Markers in Postpartum-Derived Cells

In the preceding example, similarities and differences in cells derivedfrom the human placenta and the human umbilical cord were assessed bycomparing their gene expression profiles with those of cells derivedfrom other sources (using an oligonucleotide array). Six “signature”genes were identified: oxidized LDL receptor 1, interleukin-8, rennin,reticulon, chemokine receptor ligand 3 (CXC ligand 3), and granulocytechemotactic protein 2 (GCP-2). These “signature” genes were expressed atrelatively high levels in postpartum-derived cells.

The procedures described in this example were conducted to verify themicroarray data and find concordance/discordance between gene andprotein expression, as well as to establish a series of reliable assayfor detection of unique identifiers for placenta- and umbilicus-derivedcells.

Methods & Materials Cells:

Placenta-derived cells (three isolates, including one isolatepredominately neonatal as identified by karyotyping analysis),umbilicus-derived cells (four isolates), and Normal Human DermalFibroblasts (NHDF; neonatal and adult) grown in Growth Medium withpenicillin/streptomycin in a gelatin-coated T75 flask. Mesechymal StemCells (MSCS) were grown in Mesenchymal Stem Cell Growth Medium Bulletkit (MSCGM; Cambrex, Walkerville, Md.).

For the IL-8 protocol, cells were thawed from liquid nitrogen and platedin gelatin-coated flasks at 5,000 cells/cm², grown for 48 hours inGrowth Medium and then grown for further 8 hours in 10 milliliters ofserum starvation medium [DMEM—low glucose (Gibco, Carlsbad, Calif.),penicillin/streptomycin (Gibco, Carlsbad, Calif.) and 0.1% (w/v) BovineSerum Albumin (BSA; Sigma, St. Louis, Mo.)]. After this treatment RNAwas extracted and the supernatants were centrifuged at 150×g for 5minutes to remove cellular debris. Supernatants were then frozen at −80°C. for ELISA analysis.

Cell Culture for ELISA Assay:

Postpartum cells derived from placenta and umbilicus, as well as humanfibroblasts derived from human neonatal foreskin were cultured in GrowthMedium in gelatin-coated T75 flasks. Cells were frozen at passage 11 inliquid nitrogen. Cells were thawed and transferred to 15-millilitercentrifuge tubes. After centrifugation at 150×g for 5 minutes, thesupernatant was discarded. Cells were resuspended in 4 millilitersculture medium and counted. Cells were grown in a 75 cm² flaskcontaining 15 milliliters of Growth Medium at 375,000 cells/flask for 24hours. The medium was changed to a serum starvation medium for 8 hours.Serum starvation medium was collected at the end of incubation,centrifuged at 14,000×g for 5 minutes (and stored at −20° C.).

To estimate the number of cells in each flask, 2 milliliters oftyrpsin/EDTA (Gibco, Carlsbad, Calif.) was added each flask. After cellsdetached from the flask, trypsin activity was neutralized with 8milliliters of Growth Medium. Cells were transferred to a 15 milliliterscentrifuge tube and centrifuged at 150×g for 5 minutes. Supernatant wasremoved and 1 milliliter Growth Medium was added to each tube toresuspend the cells. Cell number was estimated using a hemocytometer.

ELISA Assay:

The amount of IL-8 secreted by the cells into serum starvation mediumwas analyzed using ELISA assays (R&D Systems, Minneapolis, Minn.). Allassays were tested according to the instructions provided by themanufacturer.

Total RNA Isolation:

RNA was extracted from confluent postpartum-derived cells andfibroblasts or for IL-8 expression from cells treated as describedabove. Cells were lysed with 350 microliters buffer RLT containingbeta-mercaptoethanol (Sigma, St. Louis, Mo.) according to themanufacturer's instructions (RNeasy Mini Kit; Qiagen, Valencia, Calif.).RNA was extracted according to the manufacturer's instructions (RNeasyMini Kit; Qiagen, Valencia, Calif.) and subjected to DNase treatment(2.7 U/sample) (Sigma St. Louis, Mo.). RNA was eluted with 50microliters DEPC-treated water and stored at −80° C.

Reverse Transcription:

RNA was also extracted from human placenta and umbilicus. Tissue (30milligram) was suspended in 700 microliters of buffer RLT containing2-mercaptoethanol. Samples were mechanically homogenized and the RNAextraction proceeded according to manufacturer's specification. RNA wasextracted with 50 microliters of DEPC-treated water and stored at −80°C. RNA was reversed transcribed using random hexamers with the TaqManreverse transcription reagents (Applied Biosystems, Foster City, Calif.)at 25° C. for 10 minutes, 37° C. for 60 minutes, and 95° C. for 10minutes. Samples were stored at −20° C.

Genes identified by cDNA microarray as uniquely regulated in postpartumcells (signature genes—including oxidized LDL receptor, interleukin-8,rennin and reticulon), were further investigated using real-time andconventional PCR.

Real-Time PCR:

PCR was performed on cDNA samples using Assays-on-Demand® geneexpression products: oxidized LDL receptor (Hs00234028); rennin(Hs00166915); reticulon (Hs00382515); CXC ligand 3 (Hs00171061); GCP-2(Hs00605742); IL-8 (Hs00174103); and GAPDH (Applied Biosystems, FosterCity, Calif.) were mixed with cDNA and TaqMan Universal PCR master mixaccording to the manufacturer's instructions (Applied Biosystems, FosterCity, Calif.) using a 7000 sequence detection system with ABI Prism 7000SDS software (Applied Biosystems, Foster City, Calif.). Thermal cycleconditions were initially 50° C. for 2 min and 95° C. for 10 min,followed by 40 cycles of 95° C. for 15 sec and 60° C. for 1 min. PCRdata was analyzed according to manufacturer's specifications (UserBulletin #2 from Applied Biosystems for ABI Prism 7700 SequenceDetection System).

Conventional PCR:

Conventional PCR was performed using an ABI PRISM 7700 (Perkin ElmerApplied Biosystems, Boston, Mass., USA) to confirm the results fromreal-time PCR. PCR was performed using 2 microliters of cDNA solution,1×AmpliTaq Gold universal mix PCR reaction buffer (Applied Biosystems,Foster City, Calif.) and initial denaturation at 94° C. for 5 minutes.Amplification was optimized for each primer set. For IL-8, CXC ligand 3,and reticulon (94° C. for 15 seconds, 55° C. for 15 seconds and 72° C.for 30 seconds for 30 cycles); for rennin (94° C. for 15 seconds, 53° C.for 15 seconds and 72° C. for 30 seconds for 38 cycles); for oxidizedLDL receptor and GAPDH (94° C. for 15 seconds, 55° C. for 15 seconds and72° C. for 30 seconds for 33 cycles). Primers used for amplification arelisted in Table 10. Primer concentration in the final PCR reaction was 1micromolar except for GAPDH, which was 0.5 micromolar. GAPDH primerswere the same as real-time PCR, except that the manufacturer's TaqManprobe was not added to the final PCR reaction. Samples were run on 2%(w/v) agarose gel and stained with ethidium bromide (Sigma, St. Louis,Mo.). Images were captured using a 667 Universal Twinpack film (VWRInternational, South Plainfield, N.J.) using a focal length Polaroidcamera (VWR International, South Plainfield, N.J.).

Immunofluorescence:

PPDCs were fixed with cold 4% (w/v) paraformaldehyde (Sigma-Aldrich, St.Louis, Mo.) for 10 minutes at room temperature. One isolate each ofumbilicus- and placenta-derived cells at passage 0 (P0) (directly afterisolation) and passage 11 (P 11) (two isolates of placenta-derived, twoisolates of umbilicus-derived cells) and fibroblasts (P11) were usedImmunocytochemistry was performed using antibodies directed against thefollowing epitopes: vimentin (1:500, Sigma, St. Louis, Mo.), desmin(1:150; Sigma—raised against rabbit; or 1:300; Chemicon, Temecula,Calif.—raised against mouse), alpha-smooth muscle actin (SMA; 1:400;Sigma), cytokeratin 18 (CK18; 1:400; Sigma), von Willebrand Factor (vWF;1:200; Sigma), and CD34 (human CD34 Class III; 1:100; DAKOCytomation,Carpinteria, Calif.). In addition, the following markers were tested onpassage 11 postpartum cells: anti-human GRO alpha--PE (1:100; BectonDickinson, Franklin Lakes, N.J.), anti-human GCP-2 (1:100; Santa CruzBiotech, Santa Cruz, Calif.), anti-human oxidized LDL receptor 1 (ox-LDLR1; 1:100; Santa Cruz Biotech), and anti-human NOGA-A (1:100; SantaCruz, Biotech).

Cultures were washed with phosphate-buffered saline (PBS) and exposed toa protein blocking solution containing PBS, 4% (v/v) goat serum(Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma, St. Louis, Mo.) for 30 minutes to access intracellular antigens.Where the epitope of interest was located on the cell surface (CD34,ox-LDL R1), Triton X-100 was omitted in all steps of the procedure inorder to prevent epitope loss. Furthermore, in instances where theprimary antibody was raised against goat (GCP-2, ox-LDL R1, NOGO-A), 3%(v/v) donkey serum was used in place of goat serum throughout. Primaryantibodies, diluted in blocking solution, were then applied to thecultures for a period of 1 hour at room temperature. The primaryantibody solutions were removed and the cultures were washed with PBSprior to application of secondary antibody solutions (1 hour at roomtemperature) containing block along with goat anti-mouse IgG—Texas Red(1:250; Molecular Probes, Eugene, Oreg.) and/or goat anti-rabbitIgG—Alexa 488 (1:250; Molecular Probes) or donkey anti-goat IgG—FITC(1:150, Santa Cruz Biotech). Cultures were then washed and 10 micromolarDAPI (Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using anappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color video camera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Preparation of Cells for FACS Analysis:

Adherent cells in flasks were washed in phosphate buffered saline (PBS)(Gibco, Carlsbad, Calif.) and detached with Trypsin/EDTA (Gibco,Carlsbad, Calif.). Cells were harvested, centrifuged, and re-suspended3% (v/v) FBS in PBS at a cell concentration of 1×10 7 per milliliter.One hundred microliter aliquots were delivered to conical tubes. Cellsstained for intracellular antigens were permeabilized with Perm/Washbuffer (BD Pharmingen, San Diego, Calif.). Antibody was added toaliquots as per manufactures specifications and the cells were incubatedfor in the dark for 30 minutes at 4° C. After incubation, cells werewashed with PBS and centrifuged to remove excess antibody. Cellsrequiring a secondary antibody were resuspended in 100 microliters of 3%FBS. Secondary antibody was added as per manufactures specification andthe cells were incubated in the dark for 30 minutes at 4° C. Afterincubation, cells were washed with PBS and centrifuged to remove excesssecondary antibody. Washed cells were resuspended in 0.5 milliliters PBSand analyzed by flow cytometry. The following antibodies were used:oxidized LDL receptor 1 (sc-5813; Santa Cruz, Biotech), GROa (555042; BDPharmingen, Bedford, Mass.), Mouse IgG1 kappa, (P-4685 and M-5284;Sigma), Donkey against Goat IgG (sc-3743; Santa Cruz, Biotech.). Flowcytometry analysis was performed with FACScalibur (Becton Dickinson SanJose, Calif.).

Results

Results of real-time PCR for selected “signature” genes performed oncDNA from cells derived from human placentae, adult and neonatalfibroblasts and Mesenchymal Stem Cells (MSCs) indicate that bothoxidized LDL receptor and rennin were expressed at higher level in theplacenta-derived cells as compared to other cells. The data obtainedfrom real-time PCR were analyzed by the AACT method and expressed on alogarithmic scale. Levels of reticulon and oxidized LDL receptorexpression were higher in umbilicus-derived cells as compared to othercells. No significant difference in the expression levels of CXC ligand3 and GCP-2 were found between postpartum-derived cells and controls.The results of real-time PCR were confirmed by conventional PCR.Sequencing of PCR products further validated these observations. Nosignificant difference in the expression level of CXC ligand 3 was foundbetween postpartum-derived cells and controls using conventional PCR CXCligand 3 primers listed above.

The production of the cytokine, IL-8 in postpartum was elevated in bothGrowth Medium-cultured and serum-starved postpartum-derived cells. Allreal-time PCR data was validated with conventional PCR and by sequencingPCR products.

When supernatants of cells grown in serum-free medium were examined forthe presence of IL-8, the highest amounts were detected in media derivedfrom umbilical cells and some isolates of placenta cells (Table 10-1).No IL-8 was detected in medium derived from human dermal fibroblasts.

Placenta-derived cells were also examined for the production of oxidizedLDL receptor, GCP-2 and GROalpha by FACS analysis. Cells tested positivefor GCP-2. Oxidized LDL receptor and GRO were not detected by thismethod.

Placenta-derived cells were also tested for the production of selectedproteins by immunocytochemical analysis Immediately after isolation(passage 0), cells derived from the human placenta were fixed with 4%paraformaldehyde and exposed to antibodies for six proteins: vonWillebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscleactin, and vimentin. Cells stained positive for both alpha-smooth muscleactin and vimentin. This pattern was preserved through passage 11. Onlya few cells (<5%) at passage 0 stained positive for cytokeratin 18.

Cells derived from the human umbilical cord at passage 0 were probed forthe production of selected proteins by immunocytochemical analysis.Immediately after isolation (passage 0), cells were fixed with 4%paraformaldehyde and exposed to antibodies for six proteins: vonWillebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscleactin, and vimentin. Umbilicus-derived cells were positive foralpha-smooth muscle actin and vimentin, with the staining patternconsistent through passage 11.

Summary:

Concordance between gene expression levels measured by microarray andPCR (both real-time and conventional) has been established for fourgenes: oxidized LDL receptor 1, rennin, reticulon, and IL-8. Theexpression of these genes was differentially regulated at the mRNA levelin PPDCs, with IL-8 also differentially regulated at the protein level.The presence of oxidized LDL receptor was not detected at the proteinlevel by FACS analysis in cells derived from the placenta. Differentialexpression of GCP-2 and CXC ligand 3 was not confirmed at the mRNAlevel, however GCP-2 was detected at the protein level by FACS analysisin the placenta-derived cells. Although this result is not reflected bydata originally obtained from the microarray experiment, this may be dueto a difference in the sensitivity of the methodologies.

Immediately after isolation (passage 0), cells derived from the humanplacenta stained positive for both alpha-smooth muscle actin andvimentin. This pattern was also observed in cells at passage 11. Theseresults suggest that vimentin and alpha-smooth muscle actin expressionmay be preserved in cells with passaging, in the Growth Medium and underthe conditions utilized in these procedures. Cells derived from thehuman umbilical cord at passage 0 were probed for the expression ofalpha-smooth muscle actin and vimentin, and were positive for both. Thestaining pattern was preserved through passage 11.

Example 11 In Vitro Immunological Evaluation of Postpartum-Derived Cells

Postpartum-derived cells (PPDCs) were evaluated in vitro for theirimmunological characteristics in an effort to predict the immunologicalresponse, if any, these cells would elicit upon in vivo transplantation.PPDCs were assayed by flow cytometry for the presence of HLA-DR, HLA-DP,HLA-DQ, CD80, CD86, and B7-H2. These proteins are expressed byantigen-presenting cells (APC) and are required for the directstimulation of naïve CD4+T cells (Abbas & Lichtman, CELLULAR ANDMOLECULAR IMMUNOLOGY, 5th Ed. (2003) Saunders, Philadelphia, p. 171).The cell lines were also analyzed by flow cytometry for the expressionof HLA-G (Abbas & Lichtman, 2003, supra), CD 178 (Coumans, et al.,(1999) Journal of Immunological Methods 224, 185-196), and PD-L2 (Abbas& Lichtman, 2003, supra; Brown, et. al. (2003) The Journal of Immunology170, 1257-1266). The expression of these proteins by cells residing inplacental tissues is thought to mediate the immuno-privileged status ofplacental tissues in utero. To predict the extent to which placenta- andumbilicus-derived cell lines elicit an immune response in vivo, the celllines were tested in a one-way mixed lymphocyte reaction (MLR).

Methods & Materials Cell Culture:

Cells were cultured to confluence in Growth Medium containingpenicillin/streptomycin in T75 flasks (Corning, Corning, N.Y.) coatedwith 2% gelatin (Sigma, St. Louis, Mo.).

Antibody Staining:

Cells were washed in phosphate buffered saline (PBS) (Gibco, Carlsbad,Calif.) and detached with Trypsin/EDTA (Gibco, Carlsbad, Mo.). Cellswere harvested, centrifuged, and re-suspended in 3% (v/v) FBS in PBS ata cell concentration of 1×10⁷ per milliliter. Antibody (Table 11-1) wasadded to one hundred microliters of cell suspension as permanufacturer's specifications and incubated in the dark for 30 minutesat 4° C. After incubation, cells were washed with PBS and centrifuged toremove unbound antibody. Cells were re-suspended in five hundredmicroliters of PBS and analyzed by flow cytometry using a FACSCaliburinstrument (Becton Dickinson, San Jose, Calif.).

Mixed Lymphocyte Reaction:

Cryopreserved vials of passage 10 umbilicus-derived cells labeled ascell line A and passage 11 placenta-derived cells labeled as cell line Bwere sent on dry ice to CTBR (Senneville, Quebec) to conduct a mixedlymphocyte reaction using CTBR SOP No. CAC-031. Peripheral bloodmononuclear cells (PBMCs) were collected from multiple male and femalevolunteer donors. Stimulator (donor) allogeneic PBMC, autologous PBMC,and postpartum cell lines were treated with mitomycin C. Autologous andmitomycin C-treated stimulator cells were added to responder (recipient)PBMCs and cultured for 4 days. After incubation, [³H]-thymidine wasadded to each sample and cultured for 18 hours. Following harvest of thecells, radiolabeled DNA was extracted, and [³H]-thymidine incorporationwas measured using a scintillation counter.

The stimulation index for the allogeneic donor (SIAD) was calculated asthe mean proliferation of the receiver plus mitomycin C-treatedallogeneic donor divided by the baseline proliferation of the receiver.The stimulation index of the PPDCs was calculated as the meanproliferation of the receiver plus mitomycin C-treated postpartum cellline divided by the baseline proliferation of the receiver.

Results Mixed Lymphocyte Reaction—Placenta-Derived Cells:

Seven human volunteer blood donors were screened to identify a singleallogeneic donor that would exhibit a robust proliferation response in amixed lymphocyte reaction with the other six blood donors. This donorwas selected as the allogeneic positive control donor. The remaining sixblood donors were selected as recipients. The allogeneic positivecontrol donor and placenta-derived cell lines were treated withmitomycin C and cultured in a mixed lymphocyte reaction with the sixindividual allogeneic receivers. Reactions were performed in triplicateusing two cell culture plates with three receivers per plate (Table11-2). The average stimulation index ranged from 1.3 (plate 2) to 3(plate 1) and the allogeneic donor positive controls ranged from 46.25(plate 2) to 279 (plate 1) (Table 11-3).

Mixed Lymphocyte Reaction—Umbilicus-Derived Cells:

Six human volunteer blood donors were screened to identify a singleallogeneic donor that will exhibit a robust proliferation response in amixed lymphocyte reaction with the other five blood donors. This donorwas selected as the allogeneic positive control donor. The remainingfive blood donors were selected as recipients. The allogeneic positivecontrol donor and placenta cell lines were mitomycin C-treated andcultured in a mixed lymphocyte reaction with the five individualallogeneic receivers. Reactions were performed in triplicate using twocell culture plates with three receivers per plate (Table 11-4). Theaverage stimulation index ranged from 6.5 (plate 1) to 9 (plate 2) andthe allogeneic donor positive controls ranged from 42.75 (plate 1) to 70(plate 2) (Table 11-5).

Antigen Presenting Cell Markers—Placenta-Derived Cells:

Histograms of placenta-derived cells analyzed by flow cytometry shownegative expression of HLA-DR, DP, DQ, CD80, CD86, and B7-H2, as notedby fluorescence value consistent with the IgG control, indicating thatplacental cell lines lack the cell surface molecules required todirectly stimulate CD4+T cells.

Immunomodulating Markers—Placenta-Derived Cells:

Histograms of placenta-derived cells analyzed by flow cytometry showpositive expression of PD-L2, as noted by the increased value offluorescence relative to the IgG control, and negative expression ofCD178 and HLA-G, as noted by fluorescence value consistent with the IgGcontrol.

Antigen Presenting Cell Markers—Umbilicus-Derived Cells:

Histograms of umbilicus-derived cells analyzed by flow cytometry shownegative expression of HLA-DR, DP, DQ, CD80, CD86, and B7-H2, as notedby fluorescence value consistent with the IgG control, indicating thatumbilical cell lines lack the cell surface molecules required todirectly stimulate CD4+T cells.

Immunomodulating Cell Markers—Umbilicus-Derived Cells:

Histograms of umbilicus-derived cells analyzed by flow cytometry showpositive expression of PD-L2, as noted by the increased value offluorescence relative to the IgG control, and negative expression ofCD178 and HLA-G, as noted by fluorescence value consistent with the IgGcontrol.

Summary:

In the mixed lymphocyte reactions conducted with placenta-derived celllines, the average stimulation index ranged from 1.3 to 3, and that ofthe allogeneic positive controls ranged from 46.25 to 279. In the mixedlymphocyte reactions conducted with umbilicus-derived cell lines theaverage stimulation index ranged from 6.5 to 9, and that of theallogeneic positive controls ranged from 42.75 to 70. Placenta- andumbilicus-derived cell lines were negative for the expression of thestimulating proteins HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2, asmeasured by flow cytometry. Placenta- and umbilicus-derived cell lineswere negative for the expression of immuno-modulating proteins HLA-G andCD 178 and positive for the expression of PD-L2, as measured by flowcytometry. Allogeneic donor PBMCs contain antigen-presenting cellsexpressing HLA-DR, DQ, CD8, CD86, and B7-H2, thereby allowing for thestimulation of naïve CD4+T cells. The absence of antigen-presenting cellsurface molecules on placenta- and umbilicus-derived cells required forthe direct stimulation of naïve CD4+ T cells and the presence of PD-L2,an immunomodulating protein, may account for the low stimulation indexexhibited by these cells in a MLR as compared to allogeneic controls.

Example 12 Secretion of Trophic Factors by Postpartum-Derived Cells

The secretion of selected trophic factors from placenta- andumbilicus-derived cells was measured. Factors selected for detectionincluded: (1) those known to have angiogenic activity, such ashepatocyte growth factor (HGF) (Rosen et al. (1997) Ciba Found. Symp.212:215-26), monocyte chemotactic protein 1 (MCP-1) (Salcedo et al.(2000) Blood 96; 34-40), interleukin-8 (IL-8) (Li et al. (2003) J.Immunol. 170:3369-76), keratinocyte growth factor (KGF), basicfibroblast growth factor (bFGF), vascular endothelial growth factor(VEGF) (Hughes et al. (2004) Ann. Thorac. Surg. 77:812-8), matrixmetalloproteinase 1 (TIMP1), angiopoietin 2 (ANG2), platelet derivedgrowth factor (PDGF-bb), thrombopoietin (TPO), heparin-binding epidermalgrowth factor (HB-EGF), stromal-derived factor 1alpha (SDF-1alpha); (2)those known to have neurotrophic/neuroprotective activity, such asbrain-derived neurotrophic factor (BDNF) (Cheng et al. (2003) Dev. Biol.258; 319-33), interleukin-6 (IL-6), granulocyte chemotactic protein-2(GCP-2), transforming growth factor beta2 (TGFbeta2); and (3) thoseknown to have chemokine activity, such as macrophage inflammatoryprotein 1alpha (MIP1a), macrophage inflammatory protein 1 beta (MIP1b),monocyte chemoattractant-1 (MCP-1), Rantes (regulated on activation,normal T cell expressed and secreted), 1309, thymus andactivation-regulated chemokine (TARC), Eotaxin, macrophage-derivedchemokine (MDC), IL-8).

Methods & Materials Cell Culture:

PPDCs from placenta and umbilicus as well as human fibroblasts derivedfrom human neonatal foreskin were cultured in Growth Medium withpenicillin/streptomycin on gelatin-coated T75 flasks. Cells werecryopreserved at passage 11 and stored in liquid nitrogen. After thawingof the cells, Growth Medium was added to the cells followed by transferto a 15 milliliter centrifuge tube and centrifugation of the cells at150×g for 5 minutes. The supernatant was discarded. The cell pellet wasresuspended in 4 milliliters Growth Medium, and cells were counted.Cells were seeded at 375,000 cells/75 cm² flask containing 15milliliters of Growth Medium and cultured for 24 hours. The medium waschanged to a serum-free medium (DMEM—low glucose (Gibco), 0.1% (w/v)bovine serum albumin (Sigma), penicillin/streptomycin (Gibco)) for 8hours. Conditioned serum-free medium was collected at the end ofincubation by centrifugation at 14,000×g for 5 minutes and stored at−20° C. To estimate the number of cells in each flask, cells were washedwith PBS and detached using 2 milliliters trypsin/EDTA. Trypsin activitywas inhibited by addition of 8 milliliters Growth Medium. Cells werecentrifuged at 150×g for 5 minutes. Supernatant was removed, and cellswere resuspended in 1 milliliter Growth Medium. Cell number wasestimated using a hemocytometer.

ELISA Assay:

Cells were grown at 37° C. in 5% carbon dioxide and atmospheric oxygen.Placenta-derived cells (batch 101503) also were grown in 5% oxygen orbeta-mercaptoethanol (BME). The amount of MCP-1, IL-6, VEGF, SDF-1alpha,GCP-2, IL-8, and TGF-beta 2 produced by each cell sample was measured byan ELISA assay (R&D Systems, Minneapolis, Minn.). All assays wereperformed according to the manufacturer's instructions.

SearchLight Multiplexed ELISA Assay:

Chemokines (MIP1a, MIP1b, MCP-1, Rantes, 1309, TARC, Eotaxin, MDC, IL8),BDNF, and angiogenic factors (HGF, KGF, bFGF, VEGF, TIMP1, ANG2,PDGF-bb, TPO, HB-EGF were measured using SearchLight Proteome Arrays(Pierce Biotechnology Inc.). The Proteome Arrays are multiplexedsandwich ELISAs for the quantitative measurement of two to 16 proteinsper well. The arrays are produced by spotting a 2×2, 3×3, or 4×4 patternof four to 16 different capture antibodies into each well of a 96-wellplate. Following a sandwich ELISA procedure, the entire plate is imagedto capture chemiluminescent signal generated at each spot within eachwell of the plate. The amount of signal generated in each spot isproportional to the amount of target protein in the original standard orsample.

Results ELISA Assay:

MCP-1 and IL-6 were secreted by placenta- and umbilicus-derived cellsand dermal fibroblasts (Table 12-1). SDF-1alpha was secreted byplacenta-derived cells cultured in 5% O 2 and by fibroblasts. GCP-2 andIL-8 were secreted by umbilicus-derived cells and by placenta-derivedcells cultured in the presence of BME or 5% 02. GCP-2 also was secretedby human fibroblasts. TGF-beta2 was not detectable by ELISA assay.

SearchLight Multiplexed ELISA Assay:

TIMP1, TPO, KGF, HGF, FGF, HBEGF, BDNF, MIP1b, MCP1, RANTES, 1309, TARC,MDC, and IL-8 were secreted from umbilicus-derived cells (Tables 12-2and 12-3). TIMP1, TPO, KGF, HGF, HBEGF, BDNF, MIP1a, MCP-1, RANTES,TARC, Eotaxin, and IL-8 were secreted from placenta-derived cells(Tables 12-2 and 12-3). No Ang2, VEGF, or PDGF-bb were detected.

Summary:

Umbilicus- and placenta-derived cells secreted a number of trophicfactors. Some of these trophic factors, such as HGF, bFGF, MCP-1 andIL-8, play important roles in angiogenesis. Other trophic factors, suchas BDNF and IL-6, have important roles in neural regeneration.

Example 13 Short-Term Neural Differentiation of Postpartum-Derived Cells

The ability of placenta- and umbilicus-derived cells (collectivelypostpartum-derived cells or PPDCs) to differentiate into neural lineagecells was examined.

Methods & Materials Isolation and Expansion of Postpartum Cells:

PPDCs from placental and umbilical tissues were isolated and expanded asdescribed in Example 1.

Modified Woodbury-Black Protocol (A):

This assay was adapted from an assay originally performed to test theneural induction potential of bone marrow stromal cells (1).Umbilicus-derived cells (022803) P4 and placenta-derived cells (042203)P3 were thawed and culture expanded in Growth Media at 5,000 cells/cm²until sub-confluence (75%) was reached. Cells were then trypsinized andseeded at 6,000 cells per well of a Titretek II glass slide (VWRInternational, Bristol, Conn.). As controls, mesenchymal stem cells (P3;1F2155; Cambrex, Walkersville, Md.), osteoblasts (P5; CC2538; Cambrex),adipose-derived cells (Artecel, U.S. Pat. No. 6,555,374 B1) (P6; Donor2) and neonatal human dermal fibroblasts (P6; CC2509; Cambrex) were alsoseeded under the same conditions.

All cells were initially expanded for 4 days in DMEM/F12 medium(Invitrogen, Carlsbad, Calif.) containing 15% (v/v) fetal bovine serum(FBS; Hyclone, Logan, Utah), basic fibroblast growth factor (bFGF; 20nanograms/milliliter; Peprotech, Rocky Hill, N.J.), epidermal growthfactor (EGF; 20 nanograms/milliliter; Peprotech) andpenicillin/streptomycin (Invitrogen). After four days, cells were rinsedin phosphate-buffered saline (PBS; Invitrogen) and were subsequentlycultured in DMEM/F12 medium+20% (v/v) FBS+penicillin/streptomycin for 24hours. After 24 hours, cells were rinsed with PBS. Cells were thencultured for 1-6 hours in an induction medium which was comprised ofDMEM/F12 (serum-free) containing 200 mM butylated hydroxyanisole, 10 μMpotassium chloride, 5 milligram/milliliter insulin, 10 μM forskolin, 4μM valproic acid, and 2 μM hydrocortisone (all chemicals from Sigma, St.Louis, Mo.). Cells were then fixed in 100% ice-cold methanol andimmunocytochemistry was performed (see methods below) to assess humannestin protein expression.

Modified Woodbury-Black Protocol (B):

PPDCs (umbilicus (022803) P11; placenta (042203) P11) and adult humandermal fibroblasts (1F1853, P11) were thawed and culture expanded inGrowth Medium at 5,000 cells/cm² until sub-confluence (75%) was reached.Cells were then trypsinized and seeded at similar density as in (A), butonto (1) 24 well tissue culture-treated plates (TCP, Falcon brand, VWRInternational), (2) TCP wells+2% (w/v) gelatin adsorbed for 1 hour atroom temperature, or (3) TCP wells+20 μg/milliliter adsorbed mouselaminin (adsorbed for a minimum of 2 hours at 37° C.; Invitrogen).

Exactly as in (A), cells were initially expanded and media switched atthe aforementioned timeframes. One set of cultures was fixed, as before,at 5 days and six hours, this time with ice-cold 4% (w/v)paraformaldehyde (Sigma) for 10 minutes at room temperature. In thesecond set of cultures, medium was removed and switched to NeuralProgenitor Expansion medium (NPE) consisting of Neurobasal-A medium(Invitrogen) containing B27 (B27 supplement; Invitrogen), L-glutamine (4mM), and penicillin/streptomycin (Invitrogen). NPE medium was furthersupplemented with retinoic acid (RA; 1 μM; Sigma). This medium wasremoved 4 days later and cultures were fixed with ice-cold 4% (w/v)paraformaldehyde (Sigma) for 10 minutes at room temperature, and stainedfor nestin, GFAP, and TuJ1 protein expression (see Table 13-1).

Two Stage Differentiation Protocol:

PPDCs (umbilicus (042203) P11, placenta (022803) P11), adult humandermal fibroblasts (P11; 1F1853; Cambrex) were thawed and cultureexpanded in Growth Medium at 5,000 cells/cm² until sub-confluence (75%)was reached. Cells were then trypsinized and seeded at 2,000 cells/cm²,but onto 24 well plates coated with laminin (BD Biosciences, FranklinLakes, N.J.) in the presence of NPE media supplemented with bFGF (20nanograms/milliliter; Peprotech, Rocky Hill, N.J.) and EGF (20nanograms/milliliter; Peprotech) [whole media composition furtherreferred to as NPE+F+E]. At the same time, adult rat neural progenitorsisolated from hippocampus (P4; (062603) were also plated onto 24 welllaminin-coated plates in NPE+F+E media. All cultures were maintained insuch conditions for a period of 6 days (cells were fed once during thattime) at which time media was switched to the differentiation conditionslisted in Table 13-2 for an additional period of 7 days. Cultures werefixed with ice-cold 4% (w/v) paraformaldehyde (Sigma) for 10 minutes atroom temperature, and stained for human or rat nestin, GFAP, and TuJ1protein expression.

Multiple Growth Factor Protocol:

Umbilicus-derived cells (P11; (042203)) were thawed and culture expandedin Growth Medium at 5,000 cells/cm² until sub-confluence (75%) wasreached. Cells were then trypsinized and seeded at 2,000 cells/cm², onto24 well laminin-coated plates (BD Biosciences) in the presence of NPE+F(20 nanograms/milliliter)+E (20 nanograms/milliliter). In addition, somewells contained NPE+F+E+2% FBS or 10% FBS. After four days of“pre-differentiation” conditions, all media were removed and sampleswere switched to NPE medium supplemented with sonic hedgehog (SHH; 200nanograms/milliliter; Sigma, St. Louis, Mo.), FGF8 (100nanograms/milliliter; Peprotech), BDNF (40 nanograms/milliliter; Sigma),GDNF (20 nanograms/milliliter; Sigma), and retinoic acid (1 μM; Sigma).Seven days post medium change, cultures were fixed with ice-cold 4%(w/v) paraformaldehyde (Sigma) for 10 minutes at room temperature, andstained for human nestin, GFAP, Tull, desmin, and alpha-smooth muscleactin expression.

Neural Progenitor Co-Culture Protocol:

Adult rat hippocampal progenitors (062603) were plated as neurospheresor single cells (10,000 cells/well) onto laminin-coated 24 well dishes(BD Biosciences) in NPE+F (20 nanograms/milliliter)+E (20nanograms/milliliter).

Separately, umbilicus-derived cells (042203) P11 and placenta-derivedcells (022803) P11 were thawed and culture expanded in NPE+F (20nanograms/milliliter)+E (20 nanograms/milliliter) at 5,000 cells/cm² fora period of 48 hours. Cells were then trypsinized and seeded at 2,500cells/well onto existing cultures of neural progenitors. At that time,existing medium was exchanged for fresh medium. Four days later,cultures were fixed with ice-cold 4% (w/v) paraformaldehyde (Sigma) for10 minutes at room temperature, and stained for human nuclear protein(hNuc; Chemicon) (Table 13-1 above) to identify PPDCs.

Immunocytochemistry:

Immunocytochemistry was performed using the antibodies listed in Table131-1. Cultures were washed with phosphate-buffered saline (PBS) andexposed to a protein blocking solution containing PBS, 4% (v/v) goatserum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma) for 30 minutes to access intracellular antigens. Primaryantibodies, diluted in blocking solution, were then applied to thecultures for a period of 1 hour at room temperature. Next, primaryantibodies solutions were removed and cultures washed with PBS prior toapplication of secondary antibody solutions (1 hour at room temperature)containing blocking solution along with goat anti-mouse IgG—Texas Red(1:250; Molecular Probes, Eugene, Oreg.) and goat anti-rabbit IgG—Alexa488 (1:250; Molecular Probes). Cultures were then washed and 10micromolar DAPI (Molecular Probes) applied for 10 minutes to visualizecell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color video camera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Results Modified Woodbury-Black Protocol (A):

Upon incubation in this neural induction composition, all cell typestransformed into cells with bipolar morphologies and extended processes.Other larger non-bipolar morphologies were also observed. Furthermore,the induced cell populations stained positively for nestin, a marker ofmultipotent neural stem and progenitor cells.

Modified Woodbury-Black Protocol (B):

When repeated on tissue culture plastic (TCP) dishes, nestin expressionwas not observed unless laminin was pre-adsorbed to the culture surface.To further assess whether nestin-expressing cells could then go on togenerate mature neurons, PPDCs and fibroblasts were exposed to NPE+RA (1μM), a media composition known to induce the differentiation of neuralstem and progenitor cells into such cells (2, 3, 4). Cells were stainedfor TuJ1, a marker for immature and mature neurons, GFAP, a marker ofastrocytes, and nestin. Under no conditions was TuJ1 detected, nor werecells with neuronal morphology observed, suggesting that neurons werenot generated in the short term. Furthermore, nestin and GFAP were nolonger expressed by PPDCs, as determined by immunocytochemistry.

Two-Stage Differentiation:

Umbilicus and placenta PPDC isolates (as well as human fibroblasts androdent neural progenitors as negative and positive control cell types,respectively) were plated on laminin (neural promoting)-coated dishesand exposed to 13 different growth conditions (and two controlconditions) known to promote differentiation of neural progenitors intoneurons and astrocytes. In addition, two conditions were added toexamine the influence of GDF5, and BMP7 on PPDC differentiation.Generally, a two-step differentiation approach was taken, where thecells were first placed in neural progenitor expansion conditions for aperiod of 6 days, followed by full differentiation conditions for 7days. Morphologically, both umbilicus- and placenta-derived cellsexhibited fundamental changes in cell morphology throughout thetime-course of this procedure. However, neuronal or astrocytic-shapedcells were not observed except for in control, neural progenitor-platedconditions Immunocytochemistry, negative for human nestin, TuJ1, andGFAP confirmed the morphological observations.

Multiple Growth Factors:

Following one week's exposure to a variety of neural differentiationagents, cells were stained for markers indicative of neural progenitors(human nestin), neurons (TuJ1), and astrocytes (GFAP). Cells grown inthe first stage in non-serum containing media had different morphologiesthan those cells in serum containing (2% or 10%) media, indicatingpotential neural differentiation. Specifically, following a two stepprocedure of exposing umbilicus-derived cells to EGF and bFGF, followedby SHH, FGF8, GDNF, BDNF, and retinoic acid, cells showed long extendedprocesses similar to the morphology of cultured astrocytes. When 2% FBSor 10% FBS was included in the first stage of differentiation, cellnumber was increased and cell morphology was unchanged from controlcultures at high density. Potential neural differentiation was notevidenced by immunocytochemical analysis for human nestin, TuJ1, orGFAP.

Neural Progenitor and PPDC Co-Culture:

PPDCs were plated onto cultures of rat neural progenitors seeded twodays earlier in neural expansion conditions (NPE+F+E). While visualconfirmation of plated PPDCs proved that these cells were plated assingle cells, human-specific nuclear staining (hNuc) 4 days post-plating(6 days total) showed that they tended to ball up and avoid contact withthe neural progenitors. Furthermore, where PPDCs attached, these cellsspread out and appeared to be innervated by differentiated neurons thatwere of rat origin, suggesting that the PPDCs may have differentiatedinto muscle cells. This observation was based upon morphology underphase contrast microscopy. Another observation was that typically largecell bodies (larger than neural progenitors) possessed morphologiesresembling neural progenitors, with thin processes spanning out inmultiple directions. HNuc staining (found in one half of the cell'snucleus) suggested that in some cases these human cells may have fusedwith rat progenitors and assumed their phenotype. Control wellscontaining only neural progenitors had fewer total progenitors andapparent differentiated cells than did co-culture wells containingumbilicus or placenta PPDCs, further indicating that both umbilicus- andplacenta-derived cells influenced the differentiation and behavior ofneural progenitors, either by release of chemokines and cytokines, or bycontact-mediated effects.

Summary:

Multiple protocols were conducted to determine the short term potentialof PPDCs to differentiate into neural lineage cells. These includedphase contrast imaging of morphology in combination withimmunocytochemistry for nestin, TuJ1, and GFAP, proteins associated withmultipotent neural stem and progenitor cells, immature and matureneurons, and astrocytes, respectively. Evidence was observed to suggestthat neural differentiation occurred in certain instances in theseshort-term protocols.

Several notable observations were made in co-cultures of PPDCs withneural progenitors. This approach, using human PPDCs along with axenogeneic cell type allowed for absolute determination of the origin ofeach cell in these cultures. First, some cells were observed in thesecultures where the cell cytoplasm was enlarged, with neurite-likeprocesses extending away from the cell body, yet only half of the bodylabeled with hNuc protein. Those cells may have been human PPDCs thathad differentiated into neural lineage cells or they may have been PPDCsthat had fused with neural progenitors. Second, it appeared that neuralprogenitors extended neurites to PPDCs in a way that indicates theprogenitors differentiated into neurons and innervated the PPDCs. Third,cultures of neural progenitors and PPDCs had more cells of rat originand larger amounts of differentiation than control cultures of neuralprogenitors alone, further indicating that plated PPDCs provided solublefactors and or contact-dependent mechanisms that stimulated neuralprogenitor survival, proliferation, and/or differentiation.

Example 14 Long-Term Neural Differentiation of Postpartum-Derived Cells

The ability of umbilicus and placenta-derived cells (collectivelypostpartum-derived cells or PPDCs) to undergo long-term differentiationinto neural lineage cells was evaluated.

Methods & Materials Isolation and Expansion of PPDCs:

PPDCs were isolated and expanded as described in previous Examples.

PPDC Cell Thaw and Plating:

Frozen aliquots of PPDCs (umbilicus (022803) P11; (042203) P11; (071003)P12; placenta (101503) P7) previously grown in Growth Medium were thawedand plated at 5,000 cells/cm² in T-75 flasks coated with laminin (BD,Franklin Lakes, N.J.) in Neurobasal-A medium (Invitrogen, Carlsbad,Calif.) containing B27 (B27 supplement, Invitrogen), L-glutamine (4 mM),and Penicillin/Streptomycin (10 milliliters), the combination of whichis herein referred to as Neural Progenitor Expansion (NPE) media. NPEmedia was further supplemented with bFGF (20 nanograms/milliliter,Peprotech, Rocky Hill, N. J.) and EGF (20 nanograms/milliliter,Peprotech, Rocky Hill, N.J.), herein referred to as NPE+bFGF+EGF.

Control Cell Plating:

In addition, adult human dermal fibroblasts (P11, Cambrex, Walkersville,Md.) and mesenchymal stem cells (P5, Cambrex) were thawed and plated atthe same cell seeding density on laminin-coated T-75 flasks inNPE+bFGF+EGF. As a further control, fibroblasts, umbilicus, and placentaPPDCs were grown in Growth Medium for the period specified for allcultures.

Cell Expansion:

Media from all cultures were replaced with fresh media once a week andcells observed for expansion. In general, each culture was passaged onetime over a period of one month because of limited growth inNPE+bFGF+EGF.

Immunocytochemistry:

After a period of one month, all flasks were fixed with cold 4% (w/v)paraformaldehyde (Sigma) for 10 minutes at room temperature.Immunocytochemistry was performed using antibodies directed against Tull(BIII Tubulin; 1:500; Sigma, St. Louis, Mo.) and GFAP (glial fibrillaryacidic protein; 1:2000; DakoCytomation, Carpinteria, Calif.). Briefly,cultures were washed with phosphate-buffered saline (PBS) and exposed toa protein blocking solution containing PBS, 4% (v/v) goat serum(Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma) for 30 minutes to access intracellular antigens. Primaryantibodies, diluted in blocking solution, were then applied to thecultures for a period of 1 hour at room temperature. Next, primaryantibodies solutions were removed and cultures washed with PBS prior toapplication of secondary antibody solutions (1 hour at room temperature)containing block along with goat anti-mouse IgG—Texas Red (1:250;Molecular Probes, Eugene, Oreg.) and goat anti-rabbit IgG—Alexa 488(1:250; Molecular Probes). Cultures were then washed and 10 micromolarDAPI (Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color video camera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Results

NPE+bFGF+EGF media slows proliferation of PPDCs and alters theirmorphology Immediately following plating, a subset of PPDCs attached tothe culture flasks coated with laminin. This may have been due to celldeath as a function of the freeze/thaw process or because of the newgrowth conditions. Cells that did attach adopted morphologies differentfrom those observed in Growth Media.

Upon confluence, cultures were passaged and observed for growth. Verylittle expansion took place of those cells that survived passage. Atthis point, very small cells with no spread morphology and withphase-bright characteristics began to appear in cultures ofumbilicus-derived cells. These areas of the flask were followed overtime. From these small cells, bifurcating processes emerged withvaricosities along their lengths, features very similar to previouslydescribed PSA-NCAM+neuronal progenitors and TuJ1+immature neuronsderived from brain and spinal cord (1, 2). With time, these cells becamemore numerous, yet still were only found in clones.

Clones of Umbilicus-Derived Cells Express Neuronal Proteins:

Cultures were fixed at one month post-thawing/plating and stained forthe neuronal protein TuJ1 and GFAP, an intermediate filament found inastrocytes. While all control cultures grown in Growth Medium and humanfibroblasts and MSCs grown in NPE+bFGF+EGF medium were found to beTuJ1-/GFAP-, TuJ1 was detected in the umbilicus and placenta PPDCs.Expression was observed in cells with and without neuronal-likemorphologies. No expression of GFAP was observed in either culture. Thepercentage of cells expressing TuJ1 with neuronal-like morphologies wasless than or equal to 1% of the total population (n=3 umbilicus-derivedcell isolates tested). While not quantified, the percentage of TuJ1+cells without neuronal morphologies was higher in umbilicus-derived cellcultures than placenta-derived cell cultures. These results appearedspecific as age-matched controls in Growth Medium did not express TuJ1.

Summary:

Methods for generating differentiated neurons (based on TuJ1 expressionand neuronal morphology) from umbilicus-derived cells were developed.While expression for TuJ1 was not examined earlier than one month invitro, it is clear that at least a small population of umbilicus-derivedcells can give rise to neurons either through default differentiation orthrough long-term induction following one month's exposure to a minimalmedia supplemented with L-glutamine, basic FGF, and EGF.

Example 15 PPDC Trophic Factors for Neural Progenitor Support

The influence of umbilicus- and placenta-derived cells (collectivelypostpartum-derived cells or PPDCs) on adult neural stem and progenitorcell survival and differentiation through non-contact dependent(trophic) mechanisms was examined.

Methods & Materials Adult Neural Stem and Progenitor Cell Isolation:

Fisher 344 adult rats were sacrificed by CO₂ asphyxiation followed bycervical dislocation. Whole brains were removed intact using bonerongeurs and hippocampus tissue dissected based on coronal incisionsposterior to the motor and somatosensory regions of the brain (Paxinos,G. & Watson, C. 1997. The Rat Brain in Stereotaxic Coordinates). Tissuewas washed in Neurobasal-A medium (Invitrogen, Carlsbad, Calif.)containing B27 (B27 supplement; Invitrogen), L-glutamine (4 mM;Invitrogen), and penicillin/streptomycin (Invitrogen), the combinationof which is herein referred to as Neural Progenitor Expansion (NPE)medium. NPE medium was further supplemented with bFGF (20nanograms/milliliter, Peprotech, Rocky Hill, N.J.) and EGF (20nanograms/milliliter, Peprotech, Rocky Hill, N.J.), herein referred toas NPE+bFGF+EGF.

Following wash, the overlying meninges were removed, and the tissueminced with a scalpel. Minced tissue was collected and trypsin/EDTA(Invitrogen) added as 75% of the total volume. DNase (100 microlitersper 8 milliliters total volume, Sigma, St. Louis, Mo.) was also added.Next, the tissue/media was sequentially passed through an 18 gaugeneedle, 20 gauge needle, and finally a 25 gauge needle one time each(all needles from Becton Dickinson, Franklin Lakes, N.J.). The mixturewas centrifuged for 3 minutes at 250 g. Supernatant was removed, freshNPE+bFGF+EGF was added and the pellet resuspended. The resultant cellsuspension was passed through a 40 micrometer cell strainer (BectonDickinson), plated on laminin-coated T-75 flasks (Becton Dickinson) orlow cluster 24-well plates (Becton Dickinson), and grown in NPE+bFGF+EGFmedia until sufficient cell numbers were obtained for the studiesoutlined.

PPDC Plating:

Postpartum-derived cells (umbilicus (022803) P12, (042103) P12, (071003)P12; placenta (042203) P12) previously grown in Growth Medium wereplated at 5,000 cells/transwell insert (sized for 24 well plate) andgrown for a period of one week in Growth Medium in inserts to achieveconfluence.

Adult Neural Progenitor Plating:

Neural progenitors, grown as neurospheres or as single cells, wereseeded onto laminin-coated 24 well plates at an approximate density of2,000 cells/well in NPE+bFGF+EGF for a period of one day to promotecellular attachment. One day later, transwell inserts containingpostpartum cells were added according to the following scheme:

-   -   a. Transwell (umbilicus-derived cells in Growth Media, 200        microliters)+neural progenitors (NPE+bFGF+EGF, 1 milliliter)    -   b. Transwell (placenta-derived cells in Growth Media, 200        microliters)+neural progenitors (NPE+bFGF+EGF, 1 milliliter)    -   c. Transwell (adult human dermal fibroblasts [1 F 1853; Cambrex,        Walkersville, Md.] P12 in Growth Media, 200 microliters)+neural        progenitors (NPE+bFGF+EGF, 1 milliliter)    -   d. Control: neural progenitors alone (NPE+bFGF+EGF, 1        milliliter)    -   e. Control: neural progenitors alone (NPE only, 1 milliliter)

Immunocytochemistry:

After 7 days in co-culture, all conditions were fixed with cold 4% (w/v)paraformaldehyde (Sigma) for a period of 10 minutes at room temperatureImmunocytochemistry was performed using antibodies directed against theepitopes listed in Table 15-1. Briefly, cultures were washed withphosphate-buffered saline (PBS) and exposed to a protein blockingsolution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula,Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 30 minutes toaccess intracellular antigens. Primary antibodies, diluted in blockingsolution, were then applied to the cultures for a period of 1 hour atroom temperature. Next, primary antibodies solutions were removed andcultures washed with PBS prior to application of secondary antibodysolutions (1 hour at room temperature) containing blocking solutionalong with goat anti-mouse IgG—Texas Red (1:250; Molecular Probes,Eugene, Oreg.) and goat anti-rabbit IgG—Alexa 488 (1:250; MolecularProbes). Cultures were then washed and 10 micromolar DAPI (MolecularProbes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color video camera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Quantitative Analysis of Neural Progenitor Differentiation:

Quantification of hippocampal neural progenitor differentiation wasexamined A minimum of 1000 cells were counted per condition or if less,the total number of cells observed in that condition. The percentage ofcells positive for a given stain was assessed by dividing the number ofpositive cells by the total number of cells as determined by DAPI(nuclear) staining.

Mass Spectrometry Analysis & 2D Gel Electrophoresis:

In order to identify unique, secreted factors as a result of co-culture,conditioned media samples taken prior to culture fixation were frozendown at −80° C. overnight. Samples were then applied to ultrafiltrationspin devices (MW cutoff 30 kD). Retentate was applied to immunoaffinitychromatography (anti-Hu-albumin; IgY) (immunoaffinity did not removealbumin from the samples). Filtrate was analyzed by MALDI. The passthrough was applied to Cibachron Blue affinity chromatography. Sampleswere analyzed by SDS-PAGE and 2D gel electrophoresis.

Results PPDC Co-Culture Stimulates Adult Neural ProgenitorDifferentiation:

Following culture with umbilicus- or placenta-derived cells, co-culturedneural progenitor cells derived from adult rat hippocampus exhibitedsignificant differentiation along all three major lineages in thecentral nervous system. This effect was clearly observed after five daysin co-culture, with numerous cells elaborating complex processes andlosing their phase bright features characteristic of dividing progenitorcells. Conversely, neural progenitors grown alone in the absence of bFGFand EGF appeared unhealthy and survival was limited.

After completion of the procedure, cultures were stained for markersindicative of undifferentiated stem and progenitor cells (nestin),immature and mature neurons (TuJ1), astrocytes (GFAP), and matureoligodendrocytes (MBP). Differentiation along all three lineages wasconfirmed while control conditions did not exhibit significantdifferentiation as evidenced by retention of nestin-positive stainingamongst the majority of cells. While both umbilicus- andplacenta-derived cells induced cell differentiation, the degree ofdifferentiation for all three lineages was less in co-cultures withplacenta-derived cells than in co-cultures with umbilicus-derived cells.

The percentage of differentiated neural progenitors following co-culturewith umbilicus-derived cells was quantified (Table 15-2).Umbilicus-derived cells significantly enhanced the number of matureoligodendrocytes (MBP) (24.0% vs. 0% in both control conditions).Furthermore, co-culture enhanced the number of GFAP+ astrocytes andTuJ1+ neurons in culture (47.2% and 8.7% respectively). These resultswere confirmed by nestin staining indicating that progenitor status waslost following co-culture (13.4% vs. 71.4% in control condition 4).

Though differentiation also appeared to be influenced by adult humanfibroblasts, such cells were not able to promote the differentiation ofmature oligodendrocytes nor were they able to generate an appreciablequantity of neurons. Though not quantified, fibroblasts did however,appear to enhance the survival of neural progenitors.

Identification of Unique Compounds:

Conditioned media from umbilicus- and placenta-derived co-cultures,along with the appropriate controls (NPE media±1.7% serum, media fromco-culture with fibroblasts), were examined for differences. Potentiallyunique compounds were identified and excised from their respective 2Dgels.

Summary:

Co-culture of adult neural progenitor cells with umbilicus or placentaPPDCs results in differentiation of those cells. Results presented inthis example indicate that the differentiation of adult neuralprogenitor cells following co-culture with umbilicus-derived cells isparticularly profound. Specifically, a significant percentage of matureoligodendrocytes was generated in co-cultures of umbilicus-derivedcells. In view of the lack of contact between the umbilicus-derivedcells and the neural progenitors, this result appears to be a functionof soluble factors released from the umbilicus-derived cells (trophiceffect).

Several other observations were made. First, there were very few cellsin the control condition where EGF and bFGF were removed. Most cellsdied and on average, there were about 100 cells or fewer per well.Second, it is to be expected that there would be very littledifferentiation in the control condition where EGF and bFGF was retainedin the medium throughout, since this is normally an expansion medium.While approximately 70% of the cells were observed to retain theirprogenitor status (nestin+), about 30% were GFAP+(indicative ofastrocytes). This may be due to the fact that such significant expansionoccurred throughout the course of the procedure that contact betweenprogenitors induced this differentiation (Song, H. et al. 2002. Nature417: 29-32).

Example 16 Transplantation of Postpartum-Derived Cells

Cells derived from the postpartum umbilicus and placenta are useful forregenerative therapies. The tissue produced by postpartum-derived cells(PPDCs) transplanted into SCID mice with a biodegradable material wasevaluated. The materials evaluated were Vicryl non-woven, 35/65 PCL/PGAfoam, and RAD 16 self-assembling peptide hydrogel.

Methods & Materials Cell Culture:

Placenta- and umbilicus-derived cells were grown in Growth Medium(DMEM—low glucose (Gibco, Carlsbad Calif.), 15% (v/v) fetal bovine serum(Cat. #SH30070.03; Hyclone, Logan, Utah), 0.001% (v/v)betamercaptoethanol (Sigma, St. Louis, Mo.), penicillin/streptomycin(Gibco)) in a gelatin-coated flasks.

Sample Preparation:

One million viable cells were seeded in 15 microliters Growth Mediumonto 5 mm diameter, 2.25 mm thick Vicryl non-woven scaffolds (64.33milligrams/cc; Lot#3547-47-1) or 5 mm diameter 35/65 PCL/PGA foam(Lot#3415-53). Cells were allowed to attach for two hours before addingmore Growth Medium to cover the scaffolds. Cells were grown on scaffoldsovernight. Scaffolds without cells were also incubated in medium.

RAD16 self-assembling peptides (3D Matrix, Cambridge, Mass. under amaterial transfer agreement) was obtained as a sterile 1% (w/v) solutionin water, which was mixed 1:1 with 1×10⁶ cells in 10% (w/v) sucrose(Sigma, St Louis, Mo.), 10 mM HEPES in Dulbecco's modified medium (DMEM;Gibco) immediately before use. The final concentration of cells in RAD16hydrogel was 1×10⁶ cells/100 microliters.

Test Material (N=4/Rx)

-   -   a. Vicryl non-woven+1×10⁶ umbilicus-derived cells    -   b. 35/65 PCL/PGA foam+1×10⁶ umbilicus-derived cells    -   c. RAD 16 self-assembling peptide+1×10⁶ umbilicus-derived cells    -   d. Vicryl non-woven+1×10⁶ placenta-derived cells    -   e. 35/65 PCL/PGA foam+1×10⁶ placenta-derived cells    -   f. RAD 16 self-assembling peptide+1×10⁶ placenta-derived cells    -   g. 35/65 PCL/PGA foam    -   h. Vicryl non-woven

Animal Preparation:

The animals were handled and maintained in accordance with the currentrequirements of the Animal Welfare Act. Compliance with the above PublicLaws were accomplished by adhering to the Animal Welfare regulations (9CFR) and conforming to the current standards promulgated in the Guidefor the Care and Use of Laboratory Animals, 7th edition.

Mice (Mus Musculus)/Fox Chase SCID/Male (Harlan Sprague Dawley, Inc.,Indianapolis, Ind.), 5 Weeks of Age:

All handling of the SCID mice took place under a hood. The mice wereindividually weighed and anesthetized with an intraperitoneal injectionof a mixture of 60 milligrams/kg KETASET (ketamine hydrochloride, AvecoCo., Inc., Fort Dodge, Iowa) and 10 milligrams/kg ROMPUN (xylazine,Mobay Corp., Shawnee, Kans.) and saline. After induction of anesthesia,the entire back of the animal from the dorsal cervical area to thedorsal lumbosacral area was clipped free of hair using electric animalclippers. The area was then scrubbed with chlorhexidine diacetate,rinsed with alcohol, dried, and painted with an aqueous iodophorsolution of 1% available iodine. Ophthalmic ointment was applied to theeyes to prevent drying of the tissue during the anesthetic period.

Subcutaneous Implantation Technique:

Four skin incisions, each approximately 1.0 cm in length, were made onthe dorsum of the mice. Two cranial sites were located transversely overthe dorsal lateral thoracic region, about 5-mm caudal to the palpatedinferior edge of the scapula, with one to the left and one to the rightof the vertebral column. Another two were placed transversely over thegluteal muscle area at the caudal sacro-lumbar level, about 5-mm caudalto the palpated iliac crest, with one on either side of the midline.Implants were randomly placed in these sites in accordance with theexperimental design. The skin was separated from the underlyingconnective tissue to make a small pocket and the implant placed (orinjected for RAD16) about 1-cm caudal to the incision. The appropriatetest material was implanted into the subcutaneous space. The skinincision was closed with metal clips.

Animal Housing:

Mice were individually housed in microisolator cages throughout thecourse of the study within a temperature range of 64° F.-79° F. andrelative humidity of 30% to 70%, and maintained on an approximate 12hour light/12 hour dark cycle. The temperature and relative humiditywere maintained within the stated ranges to the greatest extentpossible. Diet consisted of Irradiated Pico Mouse Chow 5058 (Purina Co.)and water fed ad libitum.

Mice were euthanized at their designated intervals by carbon dioxideinhalation. The subcutaneous implantation sites with their overlyingskin were excised and frozen for histology.

Histology:

Excised skin with implant was fixed with 10% neutral buffered formalin(Richard-Allan Kalamazoo, Mich.). Samples with overlying and adjacenttissue were centrally bisected, paraffin-processed, and embedded on cutsurface using routine methods. Five-micron tissue sections were obtainedby microtome and stained with hematoxylin and eosin (Poly Scientific BayShore, N.Y.) using routine methods.

Results

There was minimal ingrowth of tissue into foams (without cells)implanted subcutaneously in SCID mice after 30 days. In contrast therewas extensive tissue fill in foams implanted with umbilical-derivedcells or placenta-derived cells. Some tissue ingrowth was observed inVicryl non-woven scaffolds. Non-woven scaffolds seeded with umbilicus-or placenta-derived cells showed increased matrix deposition and matureblood vessels.

Summary:

Synthetic absorbable non-woven/foam discs (5.0 mm diameter×1.0 mm thick)or self-assembling peptide hydrogel were seeded with either cellsderived from human umbilicus or placenta and implanted subcutaneouslybilaterally in the dorsal spine region of SCID mice. The resultsdemonstrated that postpartum-derived cells could dramatically increasegood quality tissue formation in biodegradable scaffolds.

Example 17 Use of Postpartum-Derived Cells in Nerve Repair

Retinal ganglion cell (RGC) lesions have been extensively used as modelsfor various repair strategies in the adult mammalian CNS. It has beendemonstrated that retrobulbar section of adult rodent RGC axons resultsin abortive sprouting (Zeng et al., 1995) and progressive death of theparent cell population (Villegas-Perez et al., 1993). Numerous studieshave demonstrated the stimulatory effects of various exogenous andendogenous factors on the survival of axotomized RGC's and regenerationof their axons (Yip and So, 2000; Fischer et al., 2001). Furthermore,other studies have demonstrated that cell transplants can be used topromote regeneration of severed nerve axons (Li et al., 2003;Ramon-Cueto et al., 2000). Thus, these and other studies havedemonstrated that cell based therapy can be utilized for the treatmentof neural disorders that affect the spinal cord, peripheral nerves,pudendal nerves, optic nerves or other diseases/trauma due to injury inwhich nervous damage can occur.

Self-assembling peptides (PuraMatrix®, U.S. Pat. Nos. 5,670,483,5,955,343, US/PCT applications US2002/0160471, WO02/062969) have beendeveloped to act as a scaffold for cell-attachment to encapsulate cellsin 3-D, plate cells in 2-D coatings, or as microcarriers in suspensioncultures. Three-dimensional cell culture has required eitheranimal-derived materials (mouse sarcoma extract), with their inherentreproducibility and cell signaling issues, or much larger syntheticscaffolds, which fail to approximate the physical nanometer-scale andchemical attributes of native ECM. RAD 16 (NH2--(RADA) 3--COOH) and KLD(NH2-(KLDL)3--COOH) are synthesized in small (RAD 16 is 5 nanometers)oligopeptide fragments that self-assemble into nanofibers on a scalesimilar to the in vivo extracellular matrix (ECM) (3D Matrix, IncCambridge, Mass.). The self-assembly is initiated by mono- or di-valentcations found in culture media or the physiological environment. In theprotocols described in this example, RAD 16 was used as a microcarrierfor the implantation of postpartum cells into the ocular defect. In thisexample, it is demonstrated that transplants of postpartum-derived cellsPPDCs) can provide efficacy in an adult rat optic nerve axonalregeneration model.

Methods & Materials Cells:

Cultures of human adult PPDCs (umbilicus and placenta) and fibroblastcells (passage 10) were expanded for 1 passage. All cells were initiallyseeded at 5,000 cells/cm² on gelatin-coated T75 flasks in Growth Mediumwith 100 Units per milliliter penicillin, 100 micrograms per milliliterstreptomycin, 0.25 micrograms per milliliter amphotericin B (Invitrogen,Carlsbad, Calif.). At passage 11 cells were trypsinized and viabilitywas determined using trypan blue staining Briefly, 50 microliters ofcell suspension was combined with 50 microliters of 0.04% w/v trypanblue (Sigma, St. Louis Mo.) and the viable cell number, was estimatedusing a hemocytometer. Cells were then washed three times in supplementfree-Leibovitz's L-15 medium (Invitrogen, Carlsbad, Calif.). Cells werethen suspended at a concentration of 200,000 cells in 25 microliters ofRAD-16 (3DM Inc., Cambridge, Mass.), which was buffered and madeisotonic as per manufacturer's recommendations. One hundred microlitersof supplement free Leibovitz's L-15 medium was added above thecell/matrix suspension to keep it wet till use. These cell/matrixcultures were maintained under standard atmospheric conditions untiltransplantation occurred. At the point of transplantation the excessmedium was removed.

Animals and Surgery:

Long Evans female rats (220-240 gram body weight) were used. Underintraperitoneal tribromoethanol anesthesia (20 milligram/100 grams bodyweight), the optic nerve was exposed, and the optic sheath was incisedintraorbitally at approximately 2 millimeters from the optic disc, thenerve was lifted from the sheath to allow complete transsection withfine scissors (Li et al., 2003). The completeness of transsection wasconfirmed by visually observing complete separation of the proximal anddistal stumps. The control group consisted of lesioned rats withouttransplants. In transplant rats cultured postpartum cells seeded inRAD-16 were inserted between the proximal and distal stumps using a pairof microforceps. Approximately 75,000 cells in RAD-16 were implantedinto the severed optic nerve. Cell/matrix was smeared into the severedcut using a pair of fine microforceps. The severed optic nerve sheathwas closed with 10/0 black monofilament nylon (Ethicon, Inc., Edinburgh,UK). Thus, the gap was closed by drawing the cut proximal and distalends of the nerve in proximity with each other.

After cell injections were performed, animals were injected withdexamethasone (2 milligrams/kilogram) for 10 days post transplantation.For the duration of the study, animals were maintained on oralcyclosporine A (210 milligrams/liter of drinking water; resulting bloodconcentration: 250-300 micrograms/liter) (Bedford Labs, Bedford, Ohio)from 2 days pre-transplantation until end of the study. Food and waterwere available ad libitum. Animals were sacrificed at either 30 or 60days post transplantation.

CTB Application:

Three days before animals were sacrificed, under anesthesia, a glassmicropipette with a 30-50 millimeter tip was inserted tangentiallythrough the sclera behind the lens, and two 4-5 microliter aliquots of a1% retrograde tracer-cholera toxin B (CTB) aqueous solution (ListBiologic, Campbell, Calif.) was injected into the vitreous. Animals wereperfused with fixative and optic nerves were collected in the samefixative for 1 hour. The optic nerves were transferred into sucroseovernight. Twenty micrometer cryostat sections were incubated in 0.1molar glycine for 30 minutes and blocked in a PBS solution containing2.5% bovine serum albumin (BSA) (Boeringer Mannheim, Mannheim, Germany)and 0.5% triton X-100 (Sigma, St. Louis, Mo.), followed by a solutioncontaining goat anti-CTB antibody (List Biologic, Campbell, Calif.)diluted 1:4000 in a PBS containing 2% normal rabbit serum (NRS)(Invitrogen, Carlsbad, Calif.), 2.5% BSA, and 2% Triton X-100 (Sigma,St. Louis, Mo.) in PBS, and incubated in biotinylated rabbit anti-goatIgG antibody (Vector Laboratories, Burlinghame, Calif.) diluted 1:200 in2% Triton-X100 in PBS for 2 hours at room temperature. This was followedby staining in 1:200 streptavidin-green (Alexa Flour 438; MolecularProbes, Eugene, Oreg.) in PBS for 2 hours at room temperature. Stainedsections were then washed in PBS and counterstained with propidiumiodide for confocal microscopy.

Histology Preparation:

Briefly, 5 days after CTB injection, rats were perfused with 4%paraformaldehyde. Rats were given 4 cubic centimeters of urethane andwere then perfused with PBS (0.1 molar) then with 4% Para formaldehyde.The spinal cord was cut and the bone removed from the head to expose thecolliculus. The colliculus was then removed and placed in 4%paraformaldehyde. The eye was removed by cutting around the outside ofthe eye and going as far back as possible. Care was given not to cut theoptic nerve that lies on the underside of the eye. The eye was removedand the muscles were cut exposing the optic nerve this was then placedin 4% paraformaldehyde.

Results Lesions Alone:

One month after retrotubular section of the optic nerve, a number ofCTB-labeled axons were identified in the nerve segment attached to theretina. In the 200 micrometers nearest the cut, axons were seen to emita number of collaterals at right angles to the main axis and terminateas a neuromatous tangle at the cut surface. In this cut between theproximal and distal stumps, the gap was observed to be progressivelybridged by a 2-3 millimeter segment of vascularized connective tissue;however, no axons were seen to advance into this bridged area. Thus, inanimals that received lesion alone no axonal growth was observed toreach the distal stump.

RAD-16 Transplantation:

Following transplantation of RAD-16 into the cut, visible ingrowth ofvascularized connective tissue was observed. However, no axonal ingrowth was observed between the proximal and distal stumps. The resultsdemonstrate that application of RAD-16 alone is not sufficient forinducing axonal regeneration in this situation.

Transplantation of Postpartum-Derived Cells:

Transplantation of postpartum-derived cells into the severed optic nervestimulated optic nerve regrowth. Some regrowth was also observed inconditions in which fibroblast cells were implanted, although this wasminimal as compared with the regrowth observed with the transplantedplacenta-derived cells. Optic nerve regrowth was observed in 4/5 animalstransplanted with placenta-derived cells, 3/6 animals transplanted withadult dermal fibroblasts and in 1/4 animals transplanted withumbilicus-derived cells. In situations where regrowth was observed, CTBlabeling confirmed regeneration of retinal ganglion cell axons, whichwere demonstrated to penetrate through the transplant area. GFAPlabeling was also performed to determine the level of glial scarring.The GFAP expression was intensified at the proximal stump with someimmunostaining being observed through the reinervated graft.

Summary:

These results demonstrate that transplanted human adultpostpartum-derived cells are able to stimulate and guide regeneration ofcut retinal ganglion cell axons.

Example 18 Use of Postpartum-Derived Cells in the Treatment of RetinitisPigmentosa

Currently no real treatment exists for blinding disorders that stem fromthe degeneration of cells in the retina. Loss of photoreceptors as aresult of apoptosis or secondary degeneration lead to progressivedeterioration of vision, and ultimately to blindness. Diseases in whichthis occurs include age-related macular degeneration (AMD) and retinitispigmentosa (RP). RP is most commonly associated with a single genemutation, which contributes to photoreceptor cell death.

The retinal photoreceptors and adjacent retinal pigment epithelium forma functional unit. The Royal College of Surgeons (RCS) rat presents witha tyrosine receptor kinase (Merkt) defect affecting outer segmentphagocytosis, leading to photoreceptor cell death. Transplantation ofretinal pigment epithelial (RPE) cells into the subretinal space of RCSrats was found to limit the progress of photoreceptor loss and preservevisual function. In this example, it is demonstrated thatpostpartum-derived cells can be used to promote photoreceptor rescue andthus preserve photoreceptors in an RCS model.

Methods & Materials Cell Transplants:

Cultures of human adult umbilicus-derived cells, placental-derived cellsand fibroblast cells (passage 10) were expanded for 1 passage. All cellswere initially seeded at 5,000 cells/cm² on gelatin-coated T75 flasks inGrowth Medium. For subsequent passages, all cells were treated asfollows. After trypsinization, viable cells were counted after trypanblue staining Briefly, 50 microliters of cell suspension was combinedwith 50 microliters of 0.04% w/v trypan blue (Sigma, St. Louis Mo.) andthe viable cell number was estimated using a hemocytometer. Cells weretrypsinized and washed three times in supplement free-DMEM: Low glucosemedium (Invitrogen, Carlsbad, Calif.). Cultures of human adultumbilicus-derived cells, placental-derived cells and fibroblast cells atpassage 11 were trypsinized and washed twice in Leibovitz's L-15 medium(Invitrogen, Carlsbad, Calif.). For the transplantation procedure,dystrophic RCS rats were anesthetized with xylazine-ketamine (1 mg/kgi.p. of the following mixture: 2.5 ml xylazine at 20 mg/ml, 5 mlketamine at 100 mg/ml, and 0.5 ml distilled water) and their headssecured by a nose bar. Cells devoid of serum were resuspended (2×10⁵cells per injection) in 2 microliters of Leibovitz, L-15 medium(Invitrogen, Carlsbad, Calif.) and transplanted using a fine glasspipette (internal diameter 75-150 micrometers) trans-scerally. Cellswere delivered into the dorso-temporal subretinal space of anesthetized3 week old dystrophic-pigmented RCS rats (total N=10/cell type).

Cells were injected unilaterally into the right eye, while the left eyewas injected with carrier medium alone (Sham control; Leibovitz's L-15medium). Viability of residual transplant cells remained at greater than95% as assessed by trypan blue exclusion at the end of the transplantsession. After cell injections were performed, animals were injectedwith dexamethasone (2 mg/kg) for 10 days post transplantation. For theduration of the study, animals were maintained on oral cyclosporine A(210 mg/L of drinking water; resulting blood concentration: 250-300micrograms/L) (Bedford Labs, Bedford, Ohio) from 2 dayspre-transplantation until end of the study. Food and water wereavailable ad libitum. Animals were sacrificed at 60 or 90 dayspostoperatively, with some animals being sacrificed at earliertimepoints for histological assessment of short-term changes associatedwith cell transplantation.

ERG Recordings:

Following overnight dark adaptation, animals were prepared for ERGrecording under dim red light, as described in (Sauve, Y. et al., 2004,Vision Res. 44: 9-18). In brief, under anesthesia (with a mixture of 150mg/kg i.p ketamine, and 10 mg/kg i.p. xylazine) the head was securedwith a stereotaxic head holder and the body temperature monitoredthrough a rectal thermometer and maintained at 38° C. using ahomeothermic blanket. Pupils were dilated using equal parts of topical2.5% phenylephrine and 1% tropicamide. Topical anesthesia with 0.75%bupivacaine was used to prevent any corneal reflexes and a drop of 0.9%saline was frequently applied on the cornea to prevent its dehydrationand allow electrical contact with the recording electrode (gold wireloop). A 25-gauge needle inserted under the scalp, between the two eyes,served as the reference electrode. Amplification (at 1-1000 Hz bandpass,without notch filtering), stimulus presentation, and data acquisitionwere provided by the UTAS-3000 system from LKC Technologies(Gaithersburg, Md.). ERGs were recorded at 60 and 90 days of age in theumbilicus-derived cell groups and at 60 days only in theplacental-derived cell and fibroblast cell groups.

Mixed a- and b-Wave Recording, Measuring Total Rod and ConeContribution:

For the quantification of dark-adapted b-waves, recordings consisted ofsingle flash presentations (10 μsec duration), repeated 3 to 5 times toverify the response reliability and improve the signal-to-noise ratio,if required. Stimuli were presented at six increasing intensities in onelog unit steps varying from −3.6 to 1.4 log candila/m² in luminance. Tominimize the potential bleaching of rods, inter-stimulus intervals wereincreased as the stimulus luminance was elevated from 10 sec at loweststimulus intensity to 2 minutes at highest stimulus intensity. Themaximum b-wave amplitude was defined as that obtained from the flashintensity series, regardless of the stimulus intensity. The true V_(max)from fitting the data with a Naka-Rushton curve was not used because ERGresponses were often erratic at higher luminance levels in dystrophicanimals and showed tendencies for depressed responses around 0.4 and 1.4log candila/m². In order to determine the age at which ERG componentswere obtained or lost, criterion amplitudes were used: 20 μV for a- andb-waves, and 10 μV for STR-like responses. The amplitude of the b-wavewas measured from the a-wave negative peak up to the b-wave positiveapex, and not up to the peak of oscillations, which can exceed theb-wave apex. In these experiments as disease progresses in this modelthe ERG's are effectively abnormal. Thus, ERG measurements are taken onthe lower end of normal visual function. This in turn can makemeasurements noisy as you reach the limits of threshold sensitivity.

Isolation of Rod and Cone Responses:

The double flash protocol, as described in (Nixon, P. J. et al., 2001,Clin Experiment Ophthalmol. 29:193-196) was used to determine theisolation of rod and cone responses. A probe flash was presented 1 secafter a conditioning flash, using a specific feature of the UTAS-3000system (LKC Technologies) with calibrated ganzfeld; assuring completerecharge of the stimulator under the conditions used. The role of theconditioning flash in the procedure was to transiently saturate rods sothat they were rendered unresponsive to the probe flash. Response to theprobe flash was taken as reflecting cone-driven activity. A rod-drivenb-wave was obtained by subtracting the cone-driven response from themixed response (obtained following presentation of a probe flash alone,i.e. not preceded by any conditioning flash).

Functional Assessment:

Physiological retinal sensitivity testing was performed to demonstrateretinal response to dim light. Animals were anesthetized with a recoverydose of urethane at 1.25 g/kg i.p. Physiological assessment in theanimals was tested post graft in animals at 90 days by recordingmultiunit extracellular activity in the superior colliculus toillumination of respective visual receptive fields, using the methoddisclosed in (Lund, R. D. et al., 2001, Proc. Natl. Acad. Sci. USA. 98:9942-9947). This procedure was repeated for 20 independent points(spaced 200 mm apart, with each step corresponding to approximately10-150 displacements in the visual field), covering the visual field.Visual thresholds were measured as the increase in intensity overbackground and maintained at 0.02 candila/m² (luminescence unit) [atleast 2.6 logarithm units below rod saturation], required for activatingunits in the superficial 200 μm of the superior colliculus. Responseparameters were compared between transplanted and sham control eyes thatreceived vehicle alone.

Histology:

Animals were sacrificed with an overdose of urethane (12.5 g/kg). Theorientation of the eye was maintained by placing a 6.0 suture throughthe superior rectus muscle prior to enucleation. After making a cornealincision, the eyes were fixed with 2.5% parafomaldehyde, 2.5%glutaraldehyde, 0.01% picric acid in 0.1 M cacodylate buffer (pH7.4)(Sigma, St. Louis, Mo.). After fixation, the cornea and lens wereremoved by cutting around the ciliary body. A small nick was made in theperiphery of the dorsal retina prior to removal of the superior rectusto assist in maintaining orientation. The retinas were then post-fixedin 1% osmium tetroxide for 1 h. After dehydration through a series ofalcohols to epoxypropane, the retinas were embedded in TAAB embeddingresin (TAAB Laboratories, Aldemarston, UK). Semi-thin sections werestained with 1% toluidine Blue in 1% borate buffer and the ultra thinsections were contrasted with uranyl acetate and lead citrate.

For Nissl staining, sections were stained with 0.75% cresyl violet(Sigma, St. Louis, Mo.) after which they were dehydrated through gradedalcohols at 70, 95 and 100% twice. They were then placed in xylene(Sigma, St. Louis, Mo.), rinsed with PBS (pH 7.4) (Invitrogen, Carlsbad,Calif.), coverslipped and mounted with DPX mountant (Sigma, St. Louis,Mo.).

Results ERG Recordings:

Animals that received umbilicus-derived cell injections exhibitedrelative preservation of visual response properties 60 and 90 dayspost-operatively (Table 18-1). The response observed in these animalswas greater than that seen with placental-derived cell, fibroblast cellor sham treated animals. Placental-derived cell transplants (n=4) at 60days showed no improvement in a-wave (20±20) versus sham controls (O),but showed some improvement in mixed b-wave (81±72) versus sham controls(1.5±2), and good improvement in cone-b-wave (50±19) versus shamcontrols (7±7), and in rod contribution (30%) versus sham controls (O).These results indicated some improvement in visual responsiveness whencompared to sham controls.

Umbilicus-derived cell-transplanted animals (n=6) demonstrated goodimprovement in all outcome measures tested at 60 days (Table 18-1),a-wave (27±11) versus sham controls (O), mixed b-wave (117±67) versussham controls (18±13), cone-b-wave (55±25) versus sham controls (28±11),and in rod contribution (49±16%) versus sham controls (6±7%).Furthermore, at 90 days, improved responses were measured in two animalstested, with measures including: a-wave (15±7) versus sham controls (O),mixed b-wave (37±15) versus sham controls (O), cone-b-wave (16±11)versus sham controls (7±5), and in rod contribution (58±39%) versus shamcontrols (0%). These results indicate that visual responsiveness wasimproved in umbilicus-derived cell transplanted animals with evidencefor photoreceptor preservation in the RCS model. Although a diminutionin responsiveness to ERG was observed in the 90 day animals tested,their preservation of visual function in comparison to sham-treatedcontrols was good.

In contrast to either umbilicus-derived or placental-derived cells,fibroblast transplantations showed no improvement in any of theparameters tested, with values less than or equal to sham-treatedcontrols.

Histology:

Following transplantation, there was no histological evidence of aninflammatory reaction and infiltrating immune cells were not observed inNissl-stained sections in the postpartum cell groups. However,fibroblast implantations resulted in animal death (n=7) and indicationsof early stage inflammatory responses. Histologically at the 90 day timepoint in the umbilicus-derived cell transplanted animals anatomicalrescue of photoreceptors was clearly demonstrated. The photoreceptorsformed a thick layer separated by a gap from the inner nuclear layer,made up of other retinal cells. By comparison, the width of the outerlayer in the sham control was, at best, a discontinuous single layer asopposed to around 5 cells thick in the grafted eye. In comparison to anormal animal this is marginally more than half the thickness ofphotoreceptor cell layers normally observed.

Functional Assessment:

Efficacy of transplants in preventing visual loss was monitored byassessment of electrophysiological responsiveness in two animals. Thethreshold sensitivity response to light was used to define the area ofvisual field rescue in sham-injected control eyes versus eyestransplanted with umbilicus-derived cells. In nondystrophic rats, visualthresholds never exceeded 0.5 log candila/m² above background. Innon-operated dystrophic rats, the thresholds are usually in themagnitude of 4 log candila/m² units. By contrast, in non-operated shaminjected dystrophic rats, the thresholds were in the order of 2.9-4.9log candila/m² units with an average threshold of 4.0 log candila/m²units, in some instances no recording could be attained. Thus, thesham-injected rats showed some highly localized functional rescue in thetemporal retina. However, the human umbilicus-derived cell transplantedrats exhibited substantially greater levels of visual preservation withthresholds ranging from 0.8 to 2.1 log candila/m² units, with an averagethreshold of 1.3 log candila/m² units.

Summary:

Transplantation of umbilicus-derived cells into dystrophic RCS rats wasshown to preserve photoreceptors. To a lesser degree this response wasalso seen following transplantation with placental-derived cellsalthough improvement was seen in a-wave responsiveness was not observed.In this degenerative model, one would expect the a-wave to disappearwithin 30 to 60 days and the b-wave to disappear within 3 months. Thus,a retained a-wave indicates that real and normal rod function ispreserved. Rod contribution to b-wave suggests abnormal rod function isstill possible. The sustained non-rod b-wave is the measure of how muchcone function is maintained, which is a real measure of vision. Thus,the level of improvement assessed both physiologically and anatomicallyfollowing umbilicus-derived cell transplantation is well defined here.ERG measurements provide an assessment of visual function afterphotoreceptor loss, indicating changes in electrical activity in theretina. However, ERG does not provide direct information as to imageforming capability. The measurement of collicular threshold sensitivityused in this study provides an indication of relative preservation ofvisual fields. The importance of this measure is based on a correlationbetween the amounts of functional rescue and anatomical preservation andthat the data collected compares with visual field perimetry testing inhumans. The transplantation has demonstrated a retardation of thedisease process in the test animals. Thus, the results presented hereindemonstrate clear evidence of functional efficacy of grafting humanPPDCs into the subretinal space, and that rescue occurs in the generalregion in which the grafted cells are located.

Example 19 Use of Postpartum Derived Cells in the Treatment of Glaucoma

Glaucoma is a group of eye diseases causing optic nerve damage. Theoptic nerve carries images from the retina, which is the specializedlight sensing tissue, to the brain so we can see. In glaucoma, eyepressure plays a role in damaging the delicate nerve fibers of the opticnerve. When a significant number of nerve fibers are damaged, blindspots develop in the field of vision. Once nerve damage and visual lossoccur, it is permanent. Most people don't notice these blind areas untilmuch of the optic nerve damage has already occurred. If the entire nerveis destroyed, blindness results. Furthermore, this damage to the opticnerve leads to damage of retinal ganglion cell elements, whichcorrespondingly lead to loss of photoreceptors and a subsequent loss ofvision. Glaucoma is a leading cause of blindness in the world,especially in older people. Thus, if a therapy can be provided, that caneither regenerate new nerve fibers or replace existing ones, thepotential to repair the degenerative process in Glaucoma exists. In thefollowing example we describe the potential of umbilicus-derived cellsto stimulate the generation of neurons and oligodendrocytes. Thegeneration of these cell types in vivo would enable replacement of lostnerve fibers.

Methods & Materials Adult Neural Stem and Progenitor Cell Isolation:

Fisher adult rats were sacrificed by CO₂ asphyxiation followed bycervical dislocation. Whole brains were removed intact using bonerongeurs and hippocampus tissue dissected based on coronal incisionsposterior to the motor and somatosensory regions of the brain (Paxinos,G. & Watson, C. 1997. The Rat Brain in Stereotaxic Coordinates). Tissuewas washed in Neurobasal-A medium (Invitrogen, Carlsbad, Calif.)containing B27 (B27 supplement; Invitrogen), L-glutamine (4 mM;Invitrogen), and penicillin/streptomycin (Invitrogen), the combinationof which is herein referred to as Neural Progenitor Expansion (NPE)medium. NPE medium was further supplemented with bFGF (20nanograms/milliliter, Peprotech, Rocky Hill, N.J.) and EGF (20nanograms/milliliter, Peprotech, Rocky Hill, N.J.), herein referred toas NPE+bFGF+EGF.

Following wash, the overlying meninges were removed, and the tissueminced with a scalpel. Minced tissue was collected and trypsin/EDTA(Invitrogen) added as 75% of the total volume. DNAse (100 microlitersper 8 milliliters total volume, Sigma, St. Louis, Mo.) was also added.Next, the tissue/media was sequentially passed through an 18 gaugeneedle, 20 gauge needle, and finally a 25 gauge needle one time each(all needles from Becton Dickinson, Franklin Lakes, N.J.). The mixturewas centrifuged for 3 minutes at 250 g. Supernatant was removed, freshNPE+bFGF+EGF was added and the pellet resuspended. The resultant cellsuspension was passed through a 40 micrometer cell strainer (BectonDickinson), plated on laminin-coated T-75 flasks (Becton Dickinson) orlow cluster 24-well plates (Becton Dickinson), and grown in NPE+bFGF+EGFmedia until sufficient cell numbers were obtained for the studiesoutlined.

PPDC Plating:

Postpartum-derived cells (umbilicus (022803) P12, (042103) P12, (071003)P12; placenta (042203) P12) previously grown in Growth Medium wereplated at 5,000 cells/transwell insert (sized for 24 well plate) andgrown for a period of one week in Growth Medium in inserts to achieveconfluence.

Adult Neural Progenitor Plating:

Neural progenitors, grown as neurospheres or as single cells, wereseeded onto laminin-coated 24 well plates at an approximate density of2,000 cells/well in NPE+bFGF+EGF for a period of one day to promotecellular attachment. One day later, transwell inserts containingpostpartum cells were added according to the following scheme:

-   -   1. Transwell (umbilicus-derived cells in Growth Media, 200        microliters)+neural progenitors (NPE+bFGF+EGF, 1 milliliter)    -   2. Transwell (placenta-derived cells in Growth Media, 200        microliters)+neural progenitors (NPE+bFGF+EGF, 1 milliliter)    -   3. Transwell (adult human dermal fibroblasts [1 F 1853; Cambrex,        Walkersville, Md.] P12 in Growth Media, 200 microliters)+neural        progenitors (NPE+bFGF+EGF, 1 milliliter)    -   4. Control: neural progenitors alone (NPE+bFGF+EGF, 1        milliliter) 5. Control: neural progenitors alone (NPE only, 1        milliliter)

Immunocytochemistry:

After 7 days in co-culture, all conditions were fixed with cold 4% (w/v)paraformaldehyde (Sigma) for a period of 10 minutes at room temperatureImmunocytochemistry was performed using antibodies directed against theepitopes listed in Table 19-1. Briefly, cultures were washed withphosphate-buffered saline (PBS) and exposed to a protein blockingsolution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula,Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 30 minutes toaccess intracellular antigens. Primary antibodies, diluted in blockingsolution, were then applied to the cultures for a period of 1 hour atroom temperature. Next, primary antibodies solutions were removed andcultures washed with PBS prior to application of secondary antibodysolutions (1 hour at room temperature) containing blocking solutionalong with goat anti-mouse IgG—Texas Red (1:250; Molecular Probes,Eugene, Oreg.) and goat anti-rabbit IgG—Alexa 488 (1:250; MolecularProbes). Cultures were then washed and 10 micromolar DAPI (MolecularProbes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color video camera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Quantitative Analysis of Neural Progenitor Differentiation:

Quantification of hippocampal neural progenitor differentiation wasexamined A minimum of 1000 cells were counted per condition or if less,the total number of cells observed in that condition. The percentage ofcells positive for a given stain was assessed by dividing the number ofpositive cells by the total number of cells as determined by DAPI(nuclear) staining.

Mass Spectrometry Analysis & 2D Gel Electrophoresis:

In order to identify unique, secreted factors as a result of co-culture,conditioned media samples taken prior to culture fixation were frozendown at −80° C. overnight. Samples were then applied to ultrafiltrationspin devices (MW cutoff 30 kD). Retentate was applied to immunoaffinitychromatography (anti-Hu-albumin; IgY) (immunoaffinity did not removealbumin from the samples). Filtrate was analyzed by MALDI. The passthrough was applied to Cibachron Blue affinity chromatography. Sampleswere analyzed by SDS-PAGE and 2D gel electrophoresis.

Results PPDC Co-Culture Stimulates Adult Neural ProgenitorDifferentiation.

Following culture with umbilicus- or placenta-derived cells, co-culturedneural progenitor cells derived from adult rat hippocampus exhibitedsignificant differentiation along all three major lineages in thecentral nervous system. This effect was clearly observed after five daysin co-culture, with numerous cells elaborating complex processes andlosing their phase bright features characteristic of dividing progenitorcells. Conversely, neural progenitors grown alone in the absence of bFGFand EGF appeared unhealthy and survival was limited.

After completion of the procedure, cultures were stained for markersindicative of undifferentiated stem and progenitor cells (nestin),immature and mature neurons (TuJ1), astrocytes (GFAP), and matureoligodendrocytes (MBP). Differentiation along all three lineages wasconfirmed while control conditions did not exhibit significantdifferentiation as evidenced by retention of nestin-positive stainingamongst the majority of cells. While both umbilicus- andplacenta-derived cells induced cell differentiation, the degree ofdifferentiation for all three lineages was less in co-cultures withplacenta-derived cells than in co-cultures with umbilicus-derived cells.

The percentage of differentiated neural progenitors following co-culturewith umbilicus-derived cells was quantified (Table 19-2).Umbilicus-derived cells significantly enhanced the number of matureoligodendrocytes (MBP) (24.0% vs. 0% in both control conditions).Furthermore, co-culture enhanced the number of GFAP+astrocytes and TuJ1+neurons in culture (47.2% and 8.7% respectively). These results wereconfirmed by nestin staining indicating that progenitor status was lostfollowing co-culture (13.4% vs. 71.4% in control condition 4).

Though differentiation also appeared to be influenced by adult humanfibroblasts, such cells were not able to promote the differentiation ofmature oligodendrocytes nor were they able to generate an appreciablequantity of neurons. Though not quantified, fibroblasts did, however,appear to enhance the survival of neural progenitors.

Identification of Unique Compounds:

Conditioned media from umbilicus- and placenta-derived co-cultures,along with the appropriate controls (NPE media±1.7% serum, media fromco-culture with fibroblasts), were examined for differences. Potentiallyunique compounds were identified and excised from their respective 2Dgels.

Summary:

Co-culture of adult neural progenitor cells with umbilicus or placentaPPDCs results in differentiation of those cells. Results presented inthis example indicate that the differentiation of adult neuralprogenitor cells following co-culture with umbilicus-derived cells isparticularly profound. Specifically, a significant percentage of matureoligodendrocytes was generated in co-cultures of umbilicus-derivedcells. In view of the lack of contact between the umbilicus-derivedcells and the neural progenitors, this result appears to be a functionof soluble factors released from the umbilicus-derived cells (trophiceffect).

Several other observations were made. First, there were very few cellsin the control condition where EGF and bFGF were removed. Most cellsdied and on average, there were about 100 cells or fewer per well.Second, it is to be expected that there would be very littledifferentiation in the control condition where EGF and bFGF was retainedin the medium throughout, since this is normally an expansion medium.While approximately 70% of the cells were observed to retain theirprogenitor status (nestin+), about 30% were GFAP+(indicative ofastrocytes). This may be due to the fact that such significant expansionoccurred throughout the course of the procedure that contact betweenprogenitors induced this differentiation (Song, H. et al. 2002. Nature417: 29-32).

The demonstration that postpartum cells derived from umbilicus tissuecan promote the differentiation of neural stem cells into neurons andoligodendrocytes suggests that the cells may promote axonal regeneration(see Example 20) or remyelination. These data suggest that postpartumcells derived from umbilicus tissue may be protective in glaucoma.Furthermore, if postpartum cells derived from umbilicus tissue areinjected in combination with neural stem cells they would have thepotential to remyelinate lost neural elements in the retina, whichsupport vision.

Example 20 Use of Postpartum-Derived Cells in Optic Nerve Repair for theTreatment of Glaucoma

Glaucoma is a group of eye diseases causing optic nerve damage. Theoptic nerve carries images from the retina, which is the specializedlight sensing tissue, to the brain so we can see. In glaucoma, eyepressure plays a role in damaging the delicate nerve fibers of the opticnerve. When a significant number of nerve fibers are damaged, blindspots develop in the field of vision. Once nerve damage and visual lossoccur, it is permanent. Most people don't notice these blind areas untilmuch of the optic nerve damage has already occurred. If the entire nerveis destroyed, blindness results. Glaucoma is a leading cause ofblindness in the world, especially in older people. Thus, if you areable to provide a therapy that can either regenerate new nerve fibers orreplace existing ones, the potential to repair the degenerative processin Glaucoma exists. The following Example demonstrates the ability ofumbilicus-derived cells to cause axonal re-growth through a severedoptic nerve head. Such growth in vivo could provide the level of axonalregeneration required to repair vision loss.

Retinal ganglion cell (RGC) lesions have been extensively used as modelsfor various repair strategies in the adult mammalian CNS. It has beendemonstrated that retrobulbar section of adult rodent RGC axons resultsin abortive sprouting (Zeng et al., 1995) and progressive death of theparent cell population (Villegas-Perez et al., 1993). Numerous studieshave demonstrated the stimulatory effects of various exogenous andendogenous factors on the survival of axotomized RGC's and regenerationof their axons (Yip and So, 2000; Fischer et al., 2001). Furthermore,other studies have demonstrated that cell transplants can be used topromote regeneration of severed nerve axons (Li et al., 2003;Ramon-Cueto et al., 2000). Thus, these and other studies havedemonstrated that cell based therapy can be utilized for the treatmentof neural disorders that affect the spinal cord, peripheral nerves,pudendal nerves, optic nerves or other diseases/trauma due to injury inwhich nervous damage can occur.

Self-assembling peptides (PuraMatrix®, U.S. Pat. Nos. 5,670,483,5,955,343, US/PCT applications US2002/0160471, WO02/062969) have beendeveloped to act as a scaffold for cell-attachment to encapsulate cellsin 3-D, plate cells in 2-D coatings, or as microcarriers in suspensioncultures. Three-dimensional cell culture has required eitheranimal-derived materials (mouse sarcoma extract), with their inherentreproducibility and cell signaling issues, or much larger syntheticscaffolds, which fail to approximate the physical nanometer-scale andchemical attributes of native ECM. RAD 16 (NH2--(RADA) 3--COOH) and KLD(NH2-(KLDL)3--COOH) are synthesized in small (RAD 16 is 5 nanometers)oligopeptide fragments that self-assemble into nanofibers on a scalesimilar to the in vivo extracellular matrix (ECM) (3D Matrix, IncCambridge, Mass.). The self-assembly is initiated by mono- or di-valentcations found in culture media or the physiological environment. In theprotocols described in this example, RAD 16 was used as a microcarrierfor the implantation of postpartum cells into the ocular defect. In thisexample, it is demonstrated that transplants of postpartum-derived cellsPPDCs) can provide efficacy in an adult rat optic nerve axonalregeneration model.

Methods & Materials

Cells. Cultures of human adult PPDCs (umbilicus and placenta) andfibroblast cells (passage 10) were expanded for 1 passage. All cellswere initially seeded at 5,000 cells/cm² on gelatin-coated T75 flasks inGrowth Medium with 100 Units per milliliter penicillin, 100 microgramsper milliliter streptomycin, 0.25 micrograms per milliliter amphotericinB (Invitrogen, Carlsbad, Calif.). At passage 11 cells were trypsinizedand viability was determined using trypan blue staining Briefly, 50microliters of cell suspension was combined with 50 microliters of 0.04%w/v trypan blue (Sigma, St. Louis Mo.) and the viable cell number, wasestimated using a hemocytometer. Cells were then washed three times insupplement free-Leibovitz's L-15 medium (Invitrogen, Carlsbad, Calif.).Cells were then suspended at a concentration of 200,000 cells in 25microliters of RAD-16 (3DM Inc., Cambridge, Mass.), which was bufferedand made isotonic as per manufacturer's recommendations. One hundredmicroliters of supplement free Leibovitz's L-15 medium was added abovethe cell/matrix suspension to keep it wet till use. These cell/matrixcultures were maintained under standard atmospheric conditions untiltransplantation occurred. At the point of transplantation the excessmedium was removed.

Animals and Surgery:

Long Evans female rats (220-240 gram body weight) were used. Underintraperitoneal tribromoethanol anesthesia (20 milligram/100 grams bodyweight), the optic nerve was exposed, and the optic sheath was incisedintraorbitally at approximately 2 millimeters from the optic disc, thenerve was lifted from the sheath to allow complete transsection withfine scissors (Li et al., 2003). The completeness of transsection wasconfirmed by visually observing complete separation of the proximal anddistal stumps. The control group consisted of lesioned rats withouttransplants. In transplant rats cultured postpartum cells seeded inRAD-16 were inserted between the proximal and distal stumps using a pairof microforceps. Approximately 75,000 cells in RAD-16 were implantedinto the severed optic nerve. Cell/matrix was smeared into the severedcut using a pair of fine microforceps. The severed optic nerve sheathwas closed with 10/0 black monofilament nylon (Ethicon, Inc., Edinburgh,UK). Thus, the gap was closed by drawing the cut proximal and distalends of the nerve in proximity with each other.

After cell injections were performed, animals were injected withdexamethasone (2 milligrams/kilogram) for 10 days post transplantation.For the duration of the study, animals were maintained on oralcyclosporine A (210 milligrams/liter of drinking water; resulting bloodconcentration: 250-300 micrograms/liter) (Bedford Labs, Bedford, Ohio)from 2 days pre-transplantation until end of the study. Food and waterwere available ad libitum. Animals were sacrificed at either 30 or 60days post transplantation.

CTB Application:

Three days before animals were sacrificed, under anesthesia, a glassmicropipette with a 30-50 millimeter tip was inserted tangentiallythrough the sclera behind the lens, and two 4-5 microliter aliquots of a1% retrograde tracer-cholera toxin B (CTB) aqueous solution (ListBiologic, Campbell, Calif.) was injected into the vitreous. Animals wereperfused with fixative and optic nerves were collected in the samefixative for 1 hour. The optic nerves were transferred into sucroseovernight. Twenty micrometer cryostat sections were incubated in 0.1molar glycine for 30 minutes and blocked in a PBS solution containing2.5% bovine serum albumin (BSA) (Boeringer Mannheim, Mannheim, Germany)and 0.5% triton X-100 (Sigma, St. Louis, Mo.), followed by a solutioncontaining goat anti-CTB antibody (List Biologic, Campbell, Calif.)diluted 1:4000 in a PBS containing 2% normal rabbit serum (NRS)(Invitrogen, Carlsbad, Calif.), 2.5% BSA, and 2% Triton X-100 (Sigma,St. Louis, Mo.) in PBS, and incubated in biotinylated rabbit anti-goatIgG antibody (Vector Laboratories, Burlinghame, Calif.) diluted 1:200 in2% Triton-X100 in PBS for 2 hours at room temperature. This was followedby staining in 1:200 streptavidin-green (Alexa Flour 438; MolecularProbes, Eugene, Oreg.) in PBS for 2 hours at room temperature. Stainedsections were then washed in PBS and counterstained with propidiumiodide for confocal microscopy.

Histology Preparation:

Briefly, 5 days after CTB injection, rats were perfused with 4%paraformaldehyde. Rats were given 4 cubic centimeters of urethane andwere then perfused with PBS (0.1 molar) then with 4% Para formaldehyde.The spinal cord was cut and the bone removed from the head to expose thecolliculus. The colliculus was then removed and placed in 4%paraformaldehyde. The eye was removed by cutting around the outside ofthe eye and going as far back as possible. Care was given not to cut theoptic nerve that lies on the underside of the eye. The eye was removedand the muscles were cut exposing the optic nerve this was then placedin 4% paraformaldehyde.

Results Lesions Alone:

One month after retrotubular section of the optic nerve, a number ofCTB-labeled axons were identified in the nerve segment attached to theretina. In the 200 micrometers nearest the cut, axons were seen to emita number of collaterals at right angles to the main axis and terminateas a neuromatous tangle at the cut surface. In this cut between theproximal and distal stumps, the gap was observed to be progressivelybridged by a 2-3 millimeter segment of vascularized connective tissue;however, no axons were seen to advance into this bridged area. Thus, inanimals that received lesion alone no axonal growth was observed toreach the distal stump.

RAD-16 Transplantation:

Following transplantation of RAD-16 into the cut, visible ingrowth ofvascularized connective tissue was observed. However, no axonal ingrowth was observed between the proximal and distal stumps. The resultsdemonstrate that application of RAD-16 alone is not sufficient forinducing axonal regeneration in this situation.

Transplantation of Postpartum-Derived Cells:

Transplantation of postpartum-derived cells into the severed optic nervestimulated optic nerve regrowth. Some regrowth was also observed inconditions in which fibroblast cells were implanted, although this wasminimal as compared with the regrowth observed with the transplantedplacenta-derived cells. Optic nerve regrowth was observed in 4/5 animalstransplanted with placenta-derived cells, 3/6 animals transplanted withadult dermal fibroblasts and in 1/4 animals transplanted withumbilicus-derived cells. In situations where regrowth was observed, CTBlabeling confirmed regeneration of retinal ganglion cell axons, whichwere demonstrated to penetrate through the transplant area. GFAPlabeling was also performed to determine the level of glial scarring.The GFAP expression was intensified at the proximal stump with someimmunostaining being observed through the reinervated graft.

Summary:

These results demonstrate that transplanted human adultpostpartum-derived cells are able to stimulate and guide regeneration ofcut retinal ganglion cell axons. Thus, an ability to promote axonalregeneration from the optic nerve has the potential to repair a primarydefect associated to vision loss with glaucoma.

Example 21 Evaluation of the Ability of Umbilicus-Derived Cells toProtect Photoreceptors in Royal College of Surgeons Rat Model of RetinalDegeneration

The central unifying aspect of disease progression in all forms ofretinal degeneration is photoreceptor cell death leading to permanentblindness. With few exceptions such as retinal trauma, this death ismediated through a process termed apoptosis or programmed cell death inall of these diseases.

In order to assess whether application of umbilicus-derived cells couldrescue photoreceptors from apoptosis, umbilicus-derived cells weretransplanted and assessed for their ability to reduce the number ofapoptotic cells in the Royal College of Surgeon's rat model of retinaldegeneration (RCS). In this model, a mutation in retinal pigmentedepithelial (RPE) cells leads to build up of the otherwise phagocytosedouter segments of photoreceptors. As a result of this buildup,photoreceptors become uncoupled from RPE and undergo apoptosis.

One of the characteristics of apoptosis is the degradation of DNA afterthe activation of Ca/Mg dependent endonucleases. This DNA cleavage leadsto strand breakage within the DNA. One accepted measure of detectingapoptosis in histological sections is called TUNEL (terminaldeoxynucleotidyl transferase biotin-dUTP nick end labeling). TUNELidentifies apoptotic cells in situ by using terminal deoxynucleotidyltransferase (TdT) to transfer biotin-dUTP to these strand breaks ofcleaved DNA. The biotin labeled cleavage sites are then detected byreaction with fluorophore-conjugated streptavidin and visualized usingan epifluorescent microscope. In this study, TUNEL labeling was used toassess the percentage of positive apoptotic photoreceptor cells relativeto total nuclei in the outer nuclear layer.

Materials and Methods Cell Transplants:

Frozen vials of previously expanded umbilicus-derived cells (passage 10)were thawed, washed with PBS, and concentrated in Dulbecco's ModifiedEagles Medium (DMEM; Invitrogen, Grand Island, N.Y.) to 20,000 cells permicroliter. Prior to this, cells were isolated/expanded andcryopreserved in Growth Medium as previously described in Example 5.

For the transplantation procedure, dystrophic RCS rats were anesthetizedwith xylazine-ketamine (1 mg/kg i.p. of the following mixture: 2.5 mlxylazine at 20 mg/ml, 5 ml ketamine at 100 mg/ml, and 0.5 ml distilledwater) and their heads secured by a nose bar. Cells were transplantedusing a fine glass pipette (internal diameter 75-150 micrometers)trans-sclerally. Cells were delivered via single injections into thedorso-temporal subretinal space of anesthetized 3 week olddystrophic-pigmented RCS rats (total N=2/group). As controls, sets ofRCS animals underwent either a sham injection procedure containing DMEMonly or were left untreated. Congenic, non-dystrophic rats were includedas a third control group to evaluate the level of normal apoptosis inhealthy eyes.

After cell injections were performed, animals were injected withdexamethasone (2 mg/kg) for 10 days post transplantation. For theduration of the study, animals were maintained on oral cyclosporine A(210 mg/L of drinking water; resulting blood concentration: 250-300micrograms/L) (Bedford Labs, Bedford, Ohio) from 2 dayspre-transplantation until end of the study. Food and water wereavailable ad libitum. Animals were sacrificed at 8 days(dystrophic-untreated, dystrophic-sham injected, dystrophic-cellinjected, congenic control) or 67 days postoperatively(dystrophic-untreated, dystrophic-cell injected) for histologicalanalysis.

Histology:

Animals were sacrificed with an overdose of urethane (12.5 g/kg). Theorientation of the eye was maintained by placing a 6.0 suture throughthe superior rectus muscle prior to enucleation. The cornea and lenswere next removed by cutting around the ciliary body. Eyes were thenfixed with Davidson's fix containing 22% (v/v) formalin, 33% (v/v)alcohol, 11% (v/v) glacial acetic acid, and 33% (v/v) water. After 24hours, eyes were removed from fix and washed/retained in PBS forsectioning.

Eye samples were oriented, embedded in OCT (Sakura, Torrence, Calif.),and cryostat sectioned (10 μm). TUNEL staining was performed using an insitu apoptosis detection kit whereby fragmented DNA was labeled with TdTand biotin-dUTP. A streptavidin-FITC system was used to visualizelabeling. Sections were counterstained with DAPI (10 μM, Invitrogen) tovisualize cell nuclei and coverslipped.

Imaging & Analysis:

Tiled images of TUNEL and DAPI stained retina were taken at 20× using ascanning stage and TURBOSCAN software (Media Cybernetics Inc, Bethesda,Md.). ImagePro Software (Media Cybernetics Inc.) was then used toquantitately analyze the area of DAPI positive nuclei and TUNEL+ nucleiin the outer nuclear layer (ONL) after calibrating images to squaremicron area. A minimum of 8 tissue sections was analyzed per eye andaveraged to generate mean+/−standard deviation values per section.TUNEL+ area was next divided by DAPI+ area to assess the percentage ofnuclei undergoing apoptosis as determined by TUNEL. Post-hoc statisticanalysis was performed to assess whether treatment with postpartum cellsderived from umbilical tissue significantly reduced the number of TUNEL+figures versus controls (untreated or sham injected). One-tailed, pairedt-tests were performed to evaluate this possibility.

Results

TUNEL+ area in the photoreceptor specific outer nuclear layer (ONL) wasassessed as a function of DAPI+ area in RCS animals that were untreated,sham injected, or umbilicus-derived cell treated at two differenttimepoints, 8 and 67 days post injection (post natal day 29 and 88respectively). Congenic-untreated controls at P29 were used to assessthe level of apoptosis in healthy retina. Results of this analysis aresummarized in Table 22-1.

At post natal day 29, congenic eyes had very few TUNEL+ FIGS. 0.2±0.2%of total DAPI+ area) in the ONL, while 16.0±2.3% of the ONL was TUNEL+in sham injected dystrophic rats. umbilicus-derived cell treatedanimals, however, exhibited a significant decrease in TUNEL+ area(6.6±0.5%, p<0.05, vs. dystrophic-sham). This decrease was not seen inthe overall DAPI+ area at this time, suggesting that changes inapoptotic figures had not had an impact on overall photoreceptor numberin the ONL at this timepoint.

In contrast, at P88, there was a significant increase in the DAPI+ areaof untreated vs. umbilicus-derived cell treated animals (p<0.05,dystrophic untreated vs. umbilicus-derived cell) (Table 22-2). Thisdifference was shadowed by differences in the percent of TUNEL+ cells(35.9±9.0% dystrophic untreated vs. 3.8%±1.4% indystrophic-umbilicus-derived cell treated) indicating that at 67 dayspost injection, there was a lasting effect on preservation ofphotoreceptors in umbilicus-derived cell treated animals.

Summary:

Decreases in TUNEL+ area, indicative of decreased apoptosis, wereobserved as early as 8 days post injection and as late as 67 daysfollowing single dose subretinal administration of umbilicus-derivedcells (20,000 cells). This difference is confirmed by an overallsignificant increase in the total area of DAPI+ cells with time inumbilicus-derived cell treated animals, indicating an overallpreservation of photoreceptors as late as post natal day 88. To brieflysummarize, umbilicus-derived cell treatment has an overall positiveeffect in the retina, decreasing the overall TUNEL+ area (and presumablenumber of cells). This effect is lasting and maintained at 67 days postinjection.

Example 22 Evaluation of the Ability of Umbilicus-Derived Cells toStimulate RPE Phagocytosis In Vitro

One of the prominent characteristics of retinal pigmented epithelialcells (RPE) is to phagocytose the shed outer segments of photoreceptorson a daily basis. This feature is well characterized and important inmaintaining a healthy retina. However, there are numerous otherfunctions of RPE that are well described and important to maintaininghomeostasis in the eye (reviewed in Strauss et al., 2005). RPE transportions, water, and metabolic products to the blood, they transportnon-endogenous factors as well as secrete endogenous factors thatmaintain the integrity of photoreceptors as well as choriocapillaris.RPE also secrete immunosuppressive agents that establish immuneprivilege in the eye. A failure of any one of these functions can leadto degeneration of the retina, loss of visual function, and eventuallyblindness.

There is considerable building evidence to suggest that defects in RPEability to perform these functions can trigger known human diseases likeRetinitis Pigmentosa and the dry form of age-related maculardegeneration (AMD) (Gal et al., 2000; Inana et al., 2005; Inana et al.,2007; Nordgaard et al, 2006; Sundelin et al., 1998; Strauss et al.,2005). There is further evidence to support that, while not the initialtrigger, such defects participate in the pathogenesis of other retinaldegenerative diseases like Stargardts, Bests, and Lebers Amaurosis(Boulton, et al., 2007).

Restoring or augmenting any of these processes may ameliorate or preventthe further deterioration of the retina by minimizing the progression ofphotoreceptor degeneration. Doing so would ultimately protectindividuals from the possibility of further blindness.

In this study, the ability of umbilicus-derived cells to augmentphagocytosis was assessed in an in vitro model that mimics properties ofthe back of the eye.

Materials and Methods Cells:

Frozen vials of previously expanded human adult RPE (ARPE-19, AmericanType Culture Collection, Manassas, Va.) or primary human RPE isolatedfrom donor cadaver eyes (National Disease Resource Interchange,Philadelphia, Pa.) were plated onto 24 well tissue culture treatedplastic plates in Growth Medium at 20,000 cells/well.

Frozen vials of previously expanded umbilicus-derived cells (passage7-10) were thawed, washed with PBS, and plated into transwells at 10,000or 30,000/well in Growth Medium (0.4 μm pore size PET track-etchedmembrane cell culture inserts, BD Falcon). The transwells were thenadded above the existing RPE cultures in the multiwell plate. Inaddition, previously expanded human dermal fibroblasts (passage 7-10,Cambrex, Walkersville, Md.) were used as a comparison toumbilicus-derived cells.

Co-cultures were maintained at 37 deg C., 5% CO₂ in incubators for 3days to allow for growth factors to be transferred across the membranebetween postpartum cells derived from umbilical tissue and RPE cells.After that time, (1) transwells were either removed and the phagocytosisassay performed, or (2) transwells were removed and well containing RPEalone were maintain for 1 or 4 more days by themselves prior toperforming the phagocytosis assay.

Phagocytosis Assay:

This assay was performed utilizing a commercially available kit (VybrantPhagocytosis Assay, V-6694, Invitrogen), designed to provide a modelsystem for quantitating the effects of drugs or other environmentalfactors on phagocytic function. Following removal of transwells, leftover media in bottom of wells was removed and 200 μl of E. coli reagentwas added to each well. This solution contained fluorescently tagged,heat inactivated E. coli prepared as described in the kit directions.The plate was placed back in the 37° C. incubator for 2-3 hours.Following incubation, plates were removed, E. coli particle solutionremoved from each well, and trypan blue (200 μl) added to each well for1 minute at room temperature to quench any undigested fluorescent E.coli. Next, trypan blue was removed and wells were washed with PBS onetime. Fresh PBS was added and ingested E. coli was quantitated bymeasuring fluorescent activity utilizing a plate reader (SpectraMax M5,Molecular Devices, limits of excitation 480 nm, emission 520 nm, cutoff515 nm). Plate reader results were normalized to media only controls andquantitatively represented as percentage of the control cell condition,RPE alone, where 100% of control would represent equal levels ofphagocytosis between compared conditions.

Results

An analysis of phagocytosis was performed assessing the ability ofumbilicus-derived cells to effect levels of phagocytosis in cadavericprimary human RPE (age 76 donor) or a human cell line (ARPE-19, ATCC).Both low passage (passage 2) primary human RPE and ARPE-19 behavedsimilarly when co-cultured with umbilicus-derived cells in thistranswell format assay (for a schematic view, see FIG. 1).Umbilicus-derived cells stimulated phagocytosis versus RPE alone in adose dependent manner after three days co-culture (10,000umbilicus-derived cells=approx doubling in amount of phagocytosedparticles; 30,000 umbilicus-derived cells greater than 2.5 timesincrease in phagocytosis versus control) (FIG. 2). These values werestatistically significant over control, hRPE alone (*p<0.05). However,when umbilicus-derived cells were removed after three days, and the RPEcultures maintained for another 4 days (7 day assay total), overalllevels of phagocytosis went down to control levels (FIG. 2).

In addition, we assessed whether other cell types could stimulatephagocytosis similar to umbilicus-derived cells. In particular weexamined expanded human dermal fibroblasts (Cambrex Biosciences,Walkersville, Md.). In contrast to umbilicus-derived cells treatment, 3day treatment with human dermal fibroblasts led to no changes inphagocytosis above control, RPE alone (FIG. 3, *p<0.05).

Summary:

A dose dependent, significant increase in phagocytosis was observedafter three days co-culture of umbilicus-derived cells with aged (76year old donor) primary human RPE or an expanded human cell lineARPE-19. The transwell assay performed here suggests that this effectwas mediated through trophic means rather than cell contact. This resultwas lost after removal of umbilicus-derived cells for a period of 4 daysfurther implicating trophic factors in the mechanism of action sincemost growth factors are labile and would not be expected to have animpact after a further four days in culture. Finally, the observedeffect of umbilicus-derived cells on RPE phagocytosis could not bereplicated using human dermal fibroblasts suggesting that the effect wascell type specific. These results suggest that umbilicus-derived cellssecrete growth factors that stimulate phagocytosis in human retinalpigmented epithelial (RPE) cells.

Example 23 Evaluation of the Ability of Umbilicus-Derived Cells toRescue Phagocytic Function In Vitro Using Cells from the RCS Loss ofFunction Mutant

The details of the RCS model of retinal degeneration are well describedearlier in this patent application. The merTK defect associated withthis model renders these rats incompetent to phagocytose the shed outersegments of photoreceptors. Previously in Example 22 we have shown theability of umbilicus-derived cells to stimulate human RPE phagocytosisin aged, but otherwise healthy RPE or similar cell lines. Here we testedwhether we could rescue this function utilizing RPE cells from the RCSrodent, a model in which the RPE have a mutation that severely limitsthis phagocytic function and has a known orthologue in humans thatcauses retinitis pigmentosa (Gal et al., 2000).

Materials and Methods Cells:

Primary rat RPE from RCS-dystrophic rats (post natal day 11) wereisolated and plated as previously described (McLaren et al. IOVS1993:34; 317-326; McLaren IOVS 1996:37; 1213-1224). They were culturedfor one week until confluent prior to co-culture with umbilicus-derivedcells. RPE from congenic, healthy rodents were harvested and utilized asa control.

Phagocytosis Assay:

Previously expanded and frozen umbilicus-derived cells (passage 7-10)were next plated on top of RCS-RPE in a similar transwell format as inExample 22. Co-cultures (in the absence of contact between the two celltypes) were maintained for 24 hours at 37 deg C., 5% CO₂. FITC-labeledphotoreceptor outer segments (POS) were next applied to cultures for 3-6hours at 37 deg C. Cultures were then analyzed under an epifluorescentmicroscope for ingestion of FITC-POS at 12.3 hours total assay time postapplication of FITC-POS.

Quantitation:

Quantitative analysis of ingested FITC-POS was performed manually aspreviously described (McLaren et al. IOVS 1993:34; 317-326; McLaren IOVS1996:37; 1213-1224). Briefly, random fields of type I RPE were taken andthe number of ingested particles counted. Control congenic (n=6)conditions were compared to dystrophic untreated (n=5) anddystrophic-umbilicus-derived cells treated (n=5) at 40× power. Groupmeans were analyzed for differences using a student's t-test (*p<0.05).Results shown were normalized to congenic controls (100%) for visualcomparison.

Results

A quantitative analysis photoreceptor outer segment phagocytosis wasperformed to evaluate the ability of umbilicus-derived cells tostimulate phagocytosis in otherwise defective RPE from the RCS rat.Normalized to congenic control levels (normal), dystrophic untreated RPEhad a drastic decrease in ability to phagocytose the FITC-POS furtherconfirming the mutation leading to loss of function in the RCS model(25.7% of control) (FIG. 4). However, after 24 hours co-culture withumbilicus-derived cells, phagocytosis was restored to normal congeniclevels in dystrophic RPE (119.2% of control, p=0.0015 vs. dystrophicuntreated).

Summary:

In this study, the ability of umbilicus-derived cells to stimulate RPEphagocytosis was assessed. Uniquely, this model allowed us to evaluatethe effect of umbilicus-derived cells trophic factors on RPE with adefect in a phagocytic gene. Further, this model allowed us to use thephysiologic photoreceptor outer segments to evaluate phagocytosis. Theseresults suggest that trophic factors secreted by umbilicus-derived cellsrescue the loss of function inherent in the RPE cells in the RCS modelof retinal degeneration. These results further implicateumbilicus-derived cells in treating a variety of retinal degenerationsassociated with phagocytic defects in RPE (Gal et al., 2000; Inana etal., 2004; Inana et al., 2007).

Example 24 Attachment of Umbilicus-Derived Cells to Aged Bruch'sMembrane: Ability to Repopulate Degenerate Retina and Sustain RPE CellSurvival or Act Like RPE in the Prevention of AMD

Aging changes occur more prominently in submacular Bruch's membrane.Thus, because AMD related changes predominate in the macular region wesought to examine the ability of umbilicus-derived cells to survive andgrow on submacular Bruch's membrane. A demonstration ofumbilicus-derived cells to attach to submacular membranes would provideevidence that these cells could survive in this environment in vivo. Theattachment of umbilicus-derived cells would provide a potential tointegrate and function like RPE or alternately provide a matrix platformon which RPE could repopulate and function.

Previous studies utilizing a Bruch's membrane explant model system showthat retinal pigment epithelial cells have limited capacity to surviveon aged Bruch's membrane, even when a robust fetal cell line is used.These results predict that RPE transplant in patients, particularlypatients with age-related macular degeneration (AMD), will not beeffective. In fact, combined RPE transplantation and choridalneovascular membrane excision has been attempted in AMD eyes, but it hasnot led to significant visual improvement in most patients. In contrast,RPE transplantation in animal models of retinal degeneration has beenproved to rescue photoreceptors and preserve visual acuity. Althoughanimal studies validate cell transplantation as a means of achievingphotoreceptor rescue, laboratory animals in which RPE transplantationhas been successful do not accurately reproduce the age-relatedmodifications of Bruch's membrane in human eyes, which may have asignificant effect on cell graft survival.

With normal aging, human Bruch's membrane, especially in the submacularregion, undergoes numerous changes (e.g., increased thickness,deposition of extracellular matrix and lipids, cross-linking of protein,non-enzymatic formation of advanced glycation end products). BMthickness appears to increase linearly with aging. Membranous debris,filamentous material, and coated vesicles accumulate primarily in theinner collagenous layer by early adulthood and continue to do sothroughout adult life and by late middle age, lipid deposition in BM isapparent. Basal laminar deposit, which comprises mostly wide-spacedcollagen and other materials including laminin, membrane-bound vesicles,and fibronectin, is present in the 7th decade during normal aging. Lipidaccumulation in BM begins to increase significantly after age 40 years.

The results of RPE cell attachment and survival on aged Bruch's membraneand similar preliminary data using putative RPE derived from embryonicstem (RPE-ES) cells indicate poor survival may not be unique to RPE.Additionally, morphology of RPE-ES indicates that some of the cells maybe dedifferentiating or trans differentiating following seeding ontoBruch's membrane. These results indicate the importance of studying thebehavior of postpartum cells derived from umbilical tissue on Bruch'smembrane to determine applicability to treating patients with AMD.

Materials and Methods

The external surface of donor human eyes was trimmed to remove muscle,connective tissue and fat, and the globes were immersed in 10% povidoneiodine briefly. This was followed by washing in BSS. This was followedby two 10-minute incubations in Dulbecco's modification of Eagle'sMedium (DMEM) containing 2.5 mg/ml amphotericin B. The anterior segment,vitreous, and the retina are dissected out. Posterior segments weretrimmed to include submacular Bruch's membrane.

The RPE were gently removed from Bruch's membrane without damaging theRPE basement membrane. After dissecting out the anterior segment,vitreous, and retina from donor eyes, submacular RPE was debrided gentlyusing a microsurgical sponge (Alcon, Fort Worth, Tex.) to create asurface with intact native RPE basement membrane.

Umbilicus-derived cells were seeded at a density of 3146 cells/mm² ontothe submacular explants. Supplemented DMEM was changed every other dayfor 1, 2, 7 or 14 days. After culture of postpartum cells derived fromumbilical tissue on submacular explants, they were fixed in 2%paraformaldehyde and 2.5% glutaraldehyde. Explants were bisectedfollowing minimum overnight fixation and then examined by lightmicroscopy or scanning electron microscopy (SEM). SEM image acquisitionwas performed on a JEOL 35C equipped with a digital image acquisitionsystem (Gatan Inc., Pleasanton, Calif.). SEM analysis focused oncomparing surface morphology of the two cells, confirming the presenceof RPE basement membrane, and determining the extent of cell coverage.

Results

Umbilicus-derived cells survived and grew to confluence on explants forup to 14 days in vitro (FIG. 5). These results demonstrated thatumbilicus-derived cells can repopulate aged Bruch's membrane and thus,providing a potential platform to support RPE repopulation oralternately a replacement for lost RPE.

Summary:

Umbilicus-derived cells can repopulate aged Bruch's membrane and thus,provide a potential platform to support RPE repopulation or alternatelya replacement for lost RPE.

Example 25 Comparison of Integrin Expression Profiles ofUmbilicus-Derived Cells with RPE

Umbilicus-derived cells and ARPE-19 were analyzed for the expression ofseveral integrins to determine if umbilicus-derived cells share similarcharacteristics to RPE.

Materials and Methods

Single cell suspensions of cultured human umbilicus-derived cells andadult retinal pigment epithelial (ARPE-19) cells were prepared bydetaching cells from culture dishes with trypsin 0.5% —EDTA solution(Invitrogen, Carlsbad, Calif.). Cells were incubated with FACSFlowBuffer (Becton Dickinson, Franklin Lakes, N.J.) containing 3% fetal calfserum (blocking solution) for 30 minutes at 4° C. with one of thefollowing mouse monoclonal antibodies (Table 25-1): α1, α2, α3, α4, α5,αvβ3, α2β1, β1 and α5β1. All washes and incubations were done inblocking solution at 4° C. Non-conjugated primary antibody labeled cellswere washed and incubated in PE-conjugated goat anti-mouse secondaryantibody for 15 minutes. Isotype controls contained cells incubated inPE-conjugated goat anti-mouse secondary antibodies (Table 25-2). Atleast 10,000 cells were analyzed with a Becton Dickinson FACSCaliburflow cytometer (Becton Dickinson, Franklin Lakes, N.J.).

Results

The results demonstrated that umbilicus-derived cells express severalintegrins associated with both fetal and adult RPE (Table 25-1). Theseresults clearly demonstrate that umbilicus-derived cells express thenatural matrix molecules that would allow them to integrate and adhereto Bruch's membrane as was demonstrated in Example 24. Furthermore, theintegrin expression is similar to that previously published for fetalRPE.

Summary:

These data suggest that umbilicus-derived cells administration to theeye may enhance the grafting of an adult RPE cell transplantation.Furthermore, the results confirm that umbilicus-derived cells expressthe relative cell surface markers necessary for them to adhere toBruch's membrane.

Example 26 Effect of Umbilicus-Derived Cell Treatment on Phagocytosis inAged Human RPE—Gene Expression Analysis

Age-related macular degeneration (AMD) affects approximately fifteenmillion people over the age of sixty and two million new cases arediagnosed each year. Those stricken by this disease experience adecrease in visual function, leading to blindness.

In this condition, both retinal pigment epithelium cells (RPE) andphotoreceptors are affected. The interaction of the RPE andphotoreceptor outer segments is crucial for the function and survival ofthe photoreceptors. Normally, RPE function to synthesize and secretecomplement factor H (CFH) as well as to ingest and degrade shedphotoreceptor outer segments. Failure of RPE in phagocytosis leads tophotoreceptor cell death, as observed in the Royal College of Surgeons(RCS) rat (Dowling and Sidman, 1962). In the RCS model, a mutation inthe gene Mertk results in the normal binding of photoreceptor outersegments but the inability to ingest shed tips of photoreceptor outersegments. As oxidized shed segments build up, synthesis and secretion ofCFH by RPE is affected. The role of CFH is to regulate complementactivation. If inhibited, CFH-mediated protection of RPE cells may bereduced thus leading to the onset of AMD (Chen, 2007).

In order to assess the impact umbilicus-derived cell application has onhuman aged RPE, differences in host gene expression were examined Humanaged RPE were used in this study for the following reasons: [1] AMDgenerally affects people over the age of sixty and [2] evidence suggeststhat with age, lipofuscin build-up occurs. Lipofuscin build-up in thelysosomal compartment of RPE may cause insufficient synthesis of CFHthus resulting a lack of protection against complement damage.Therefore, a panel of genes associated with phagocytosis, proteosomaldegradation and inflammation was established and subsequent geneexpression levels were compared.

Materials and Methods

Human Retinal Pigmented Epithelium (hRPE) Isolation:

The hRPE was dissected from one set of human eyes (75 year old Caucasianfemale). The donor had no known diagnosed eye disease. However, afterexamining the gross appearance of the retina and RPE, small retinalhemorrhages were noted (hallmark characteristic of those affected byAMD). The eyes were obtained through the National Disease ResearchInterchange (NDRI). After removing the sclera from both eyes, the hRPEwas dissected away from the retina. The hRPE was cut into pieces andenzymatically digested for 30 minutes in a solution containing trypsin(JRH Bioscience), kynurenic acid ([20 mg], Sigma) and vitrase ([200units], ISTA Pharmaceuticals). To further disassociate the tissue, cellswere triturated and then briefly vortexed. To stop the digestionprocess, DMEM—low glucose media (Invitrogen, Carlsbad, Calif.)containing 15 percent (v/v) fetal bovine serum (FBS; Hyclone, Logan,Utah) was added. After that cells were forced through a 100 μm nyloncell strainer (BD Falcon). The resulting solution was then centrifugedfor 5 minutes at 250×g. The supernatant was removed and the cells wereresuspended in complete medium containing DMEM—low glucose (Invitrogen,Carlsbad, Calif.), 15 percent (v/v) fetal bovine serum (FBS; Hyclone,Logan, Utah), 0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis, Mo.),and penicillin/streptomycin (5,000 Units/mL). Cells were counted with ahemocytometer and plated (concentration of 1×105 cells/well) into asix-well laminin coated plate (BD Biosciences). The plate was thenplaced into a 37° C. incubator.

Preparation of Umbilicus-Derived Cells and Co-Culture Assay:

Frozen aliquots of previously expanded umbilicus-derived cells (passage7) were thawed, washed, counted and seeded (5×10⁴ cells/inserts) intoeither a 6-well cell culture plate (Corning) or into cell cultureinserts (pore size: 0.4 μm, BD Biosciences, Franklin Lakes, N.J.). Allcells were grown in the same complete medium as hRPE. Theumbilicus-derived cells inserts were placed directly on top of threewells containing hRPE. The plates were then placed into a 37° C.incubator for 3 days.

RNA Isolation:

After 3 days, the inserts were removed and all cells (hRPE andumbilicus-derived cells) were trypsinized, centrifuged, resuspended andcounted. The untreated hRPE and treated hRPE were lysed by addition of350 μL RLT Buffer (RNAeasy Mini kit, QIAGEN). The lysate was appliedonto a QIAshredder spin column and placed in a 2 mL collection tube andthen centrifuged for 2 minutes at maximum speed (18,000 g; Microfuge 18Centrifuge, Beckman-Coulter Cat#367160). One volume of 70% ethanol (200proof, Sigma, Cat # E7023-500ML) was added to the homogenized lysate andapplied to an RNeasy mini column placed in a 2 mL collection tube(supplied). The column was centrifuged for 15 seconds at greater than orequal to 8000×g (greater than or equal to 10,000 rpm). The flow throughwas discarded. Buffer RW1 (700 μl) was added to the RNeasy column andcentrifuged for 15 seconds at greater than or equal to 8000×g (greaterthan or equal to 10,000 rpm) to wash the column. The flow through wasdiscarded. The RNeasy column was transferred into a new 2 mL collectiontube (supplied) and 5000 μL Buffer RPE was applied to the RNeasy column.The column was centrifuged for 15 seconds at greater than or equal to8000×g (greater than or equal to 10,000 rpm). The flow through wasdiscarded. Another 5000 μL of Buffer RPE was applied to the RNeasycolumn and centrifuge for 2 minutes at greater than or equal to 8000×g(greater than or equal to 10,000 rpm) to dry the RNeasy column. TheRNeasy column was transferred to a new 1.8 mL collection tube (DNALoBind tube 1.5 mL 22 43 102-1, Eppendorf AG) and centrifuged in amicrocentrifuge at full speed for 1 minute. To elute the RNA from theRNeasy column, the column was transferred to a new 1.5 mL collectiontube (supplied) and 30 μL RNase-free water was applied. The tube wasthen centrifuged for 1 minute at greater than or equal to 8000×g(greater than or equal to 10,000 rpm). After centrifugation, the tubewas removed, capped and labeled and then transferred to a −80° C.freezer.

RNA Quality and Quantification:

The quantity and quality of RNA was determined using SOFTMAX Prosoftware on the SPECTRAMAX M5 spectrophotometer (Molecular Devices).Deionized water (dH20) was used as a blank and was loaded into the firstwell of a 96-well UV plate (Costar, Corning). Diluted RNA samples (1:20)were loaded into subsequent wells and the reading was taken. The RNAconcentration was calculated: (μg/mL)=(OD260)×(dilution factor)×(40 μgRNA/ml). Then 10 μL of each diluted RNA sample was loaded into anagarose E-gel (Invitrogen) along with 10 μL of Trackh (50 bp DNA ladder,Invitrogen). Samples were run for 15 minutes. After time had expired,images were taken of the gel using the Chemi Doc XRS camera system(Bio-Rad).

First Strand cDNA Synthesis:

The synthesis of cDNA was performed using the SUPERSCRIPT IIIFirst-Strand Synthesis System (Invitrogen, Carlsbad, Calif., USA).Reagents were combined and added to each tube as shown in Table 26-1.

Tubes were then transferred to a Thermal Cycler (Bio-Rad Cat#170-9703),incubated at 65° C. for 5 minutes and then cooled to 4° C. Next, 20 μLof cDNA Synthesis Mix (Table 26-2) was added to each tube. The tubeswere then heated to 50° C. for 50 minutes, followed by another 85° C.cycle, which lasted for 5 minutes, and finally a cooling 4° C. cycle for15 minutes. At this time, 1 μL of RNase H was added to each tube. Thetubes were again incubated for 20 minutes at 37° C. At the end of thiscycle, the cDNA synthesis reaction was used immediately for RT-PCR.

Real-Time Quantitative RT-PCR:

The mRNA levels of the analyzed genes were quantified by real timeRT-PCR. Master mix was made (Table 26-3) and then pipetted into a FastThermal Cycler Optical 96-well plate (Applied Biosystems).

The RT-PCR reaction was performed using the 7900HT Fast Real-Time PCRSystem (Applied Biosystems). Thermal cycle conditions were as follows:50° C. for 2 minutes, 95° C. for 10 minutes followed by 40 cycles of 95°C. for 15 seconds and 60° C. for 1 minute. At the completion of thereaction, the data was saved and then analyzed with the SDS 2.2.2 systemsoftware (Applied Biosystems).

Results Genes Used for Analysis:

The names of the genes that were chosen for this analysis, theirabbreviation, catalogue number and proposed function in the eye aresummarized in Table 26-4. The genes were chosen based on theirimportance in functions for RPE.

Real Time RT-PCR:

For the calibration of mRNA expression levels, GAPDH was selected toserve as the endogenous control. Thus for analysis, the expressionlevels of all genes were normalized to GAPDH.

In this analysis, the gene expression levels of untreated RPE werecompared to umbilicus-derived cell treated RPE (Table 26-5). In hRPEco-cultured with umbilicus-derived cells, increases expression in thefollowing genes were observed: MERTK, CRALBP, INTAV, CATHD and CFH.(2.8±0.2 co-culture vs. 8.2±0.2 control, *p<0.05). These increases wereseen only after 3 days of co-culture with umbilicus-derived cells. Theseresults suggest that umbilicus-derived cell treatment had an impact onthe expression of genes associated with phagocytosis, proteosomaldegradation and inflammation.

Summary:

Current physical treatments fail to improve visual function for thoseaffected by AMD. Retinal repair may require the transplantation ofhealthy cells for the treatment of this disease. Co-culture ofumbilicus-derived cells on aged hRPE has shown to impact the expressionof genes associated with phagocytosis, proteosomal degradation andinflammation.

Accumulating evidence suggests that immunological factors, such as CFH,may play an important role in the development of macular degenerationdevelopment. It has been shown in vitro that freshly plated human RPEcells express high levels of CFH. However, when human RPE cells areexposed to prolonged incubation with oxidized photoreceptors rod outersegments or with pro-inflammatory cytokines such as, TNF-alpha and IL-6,CFH is down regulated (M. Chen et al, 2006). This study suggests thatthe co-culture of umbilicus-derived cells could be used for treatment ofmacular degeneration as well as other ocular indications wherephagocytosis or inflammation may be affected.

Example 27 Gene Profiling Approach to Identify Potential Mechanisms bywhich Umbilicus-Derived Cells Preserve Vision in the Royal College ofSurgeons Rat Model of Retinal Degeneration Materials and Methods CellCulture for Trophic Factor Secretion:

Umbilicus-derived cells (population doubling 20) was plated at a seedingdensity of 10,000/cm² into gelatin-coated 6-well plates in Growth Mediaas described in the previous example. After 24 hrs, the culture mediawas replaced with 1 ml fresh growth media. The supernatant of theculture was collected at day 1, 3, 7 and saved frozen at −80° C. Cellnumber counts were performed for each individual sample. These culturemedia were sent to Pierce Biotechnology Inc. (Worcester, Mass.) for theanalysis described below.

ELISA:

ELISA for HGF and IGF followed the instruction from R&D systems.Briefly, working standards were prepared as directed in the instructionmanual (HGF: 125 pg/ml-4000 pg/ml; IGF: 94 pg/ml-6000 pg/ml). Standards,control, or sample (50 μl) were added to each well and were incubatedfor 2 hours at room temperature for HGF and at 4° C. for IGF. The platewas washed four times. 200 μL of HGF or IGF conjugate was added to eachwell and incubated for 1.75 hours at room temperature for HGF and for 1hour at 4° C. for IGF. The plates were washed for 4 times, then 200 μLof Substrate Solution was added to each well and incubated for 30minutes at room temperature for HGF and IGF. 50 μL of stop solution wasadded to each well. The optical density of each well was determinedusing a microplate reader set to 450 nm.

Cell Transplants.

The transplantation procedure has been described above. Briefly, 20,000umbilicus-derived cells in 2 μl PBS were injected into thedorso-temporal subretinal space of anesthetized 3-week olddystrophic-pigmented RCS rats. As controls, sets of RCS animalsunderwent either a sham injection procedure containing PBS only, or wereleft untreated.

After cell injections were performed, animals were injected withdexamethasone (2 mg/kg) for 10 days post transplantation. For theduration of the study, animals were maintained on oral cyclosporine A(210 mg/L of drinking water; resulting blood concentration: 250-300micrograms/L) (Bedford Labs, Bedford, Ohio) from 2 dayspre-transplantation until end of the study. Food and water wereavailable ad libitum. Animals were sacrificed at day 7, 30 and 60 posttransplantation (dystrophic-untreated, dystrophic-cell injected) formicroarray study. Sham-operated animals were only collected at day 7post injection

RNA Sample Collection.

Animals were sacrificed at day 7, day 30- and day 60-post celltransplantation, with an overdose of urethane (12.5 g/kg). Eyes wereextracted from the animals and fixed in RNALater to prevent RNAdegradation. RNA extraction was performed following Invitrogen Trizolprotocol. RNA concentration and quality were examined by BIO-RADExperion Bioanalyzer. From each sample, 2 μg total RNA was used forAffimetrix rat 230 version 2 chips. Three sets of comparisons were done:day 7 treated vs. sham; day 30 treated vs. non-treated; day 60 treatedvs. non-treated. In each group, three individual samples were used.

Microarray Analysis:

The RNA samples were sent to Johnson & Johnson Pharmaceutical Researchand Development in La Jolla, Calif. where the Affymetrix rat chip wasused for microarray analysis. The raw microarray data was normalizedacross the 16 chips using Quantile-Quantile normalization. ANOVA andMultiple test correction (FDR; p-value<=0.01), two methods provided inthe Partek Pro (St. Louis, Mo.) software, were used to determinedifferences between treatment and time factors after normalization.Differentially expressed genes were identified for each time point usingthe t-test method (p-value less than or equal to 0.05) in conjunctionwith fold changes (greater than or equal to 1.5) between the mean valuesof each group. With differentially expressed genes determined, relevantpathway analysis was constructed using Ingenuity systems (IPA) fromIngenuity Systems (Redwood City, Calif.).

Results Gene Profiling Revealed Protected Photoreceptor Function:

In order to understand the possible mechanisms resulting in thepreserved visual function in the umbilicus-derived cell treated eyes, weconducted a microarray study to identify the differentially expressedgenes and the canonical pathways in the treated versus non-treated rateyes to guide further in vitro studies. This study was designed torecognize differential rat gene expression and pathway networks inresponse to umbilicus-derived cell treatment in the eyes. Total RNA wasobtained from treated and non-treated rat eyes at different time points(Days 7, 30 and 60 post cell transplantation, n=3). At each time point,the non-treated fellow eyes were used as controls. Eyes fromsham-operated rats were collected at day 7 and were used as controls forDay 7 time point. Standard hybridization procedures were performed forAffimetrix Rat 230 chip. 23,000 gene expression data were obtained fromthe Affimetrix Rat 230 chip. Raw data were normalized across all chips.987 genes were differentially expressed in treated eyes vs. controlswith statistic significance (p<0.05). Hierarchical clustering wasperformed and the heat map was generated. The heat map demonstrated thatindividual sample at the same time point tends to cluster together,suggesting an evident time effect for the treatment and at the same timepoint, treatment groups differ from the control groups.

Based on the analysis described above, different predominating genenetworks were identified at different time points after celltransplantation. At day 7, in the analysis of umbilicus-derivedcell-treated vs. sham-operated eyes, genes involved in cellproliferation and growth such as FOS, CSNK1D, and RPS6KA2 were upregulated in cell-treated eyes, as shown in Table 27-1. FOS is atranscription factor that regulates cell proliferation in many celltypes. Increased FOS expression has been documented in RPE cells duringproliferation. FOS activation is regulated by MAP kinase cascade, whichlinks the growth factor receptor activation at cell surface to thetranscription factor such as FOS in the nucleus. RPS6KA2, also known aspp90^(rsk), is a kinase involved in the activation of FOS in the MAPKcascade.

The increased expression of a kinase cascade component-pp90^(rsk) and adown-stream transcription factor—FOS strongly suggests the impact ofumbilicus-derived cells on cell proliferation in the RCS rat eyes. RPEcell proliferation potentially can increase RPE cell number in theretina and potentially enhance phagocytosis, thus, protectphotoreceptors from apoptosis, and preserve visual function.

More over, genes involved in insulin-like growth factor (IGF) pathway,such as IGFBP5, IGF2BP3, were also up-regulated as shown in Table 27-1.A wide range of biological processes are modulated by IGF-1 signalingpathway, including, for example, cell proliferation, tissue-specificdifferentiation, and protection against apoptosis. In RPE cells,activation of IGF cascade is related with reduction of apoptosis-inducedby hydrogen peroxide IGF pathway members such as IGF1 binding protein(IGFBP)5 and IGFBP3 are well documented to inhibit apoptosis in multiplecell types, including RPE cells. IGFBP5 and IGFBP3 promote theactivation of IGF1 receptor by carrying IGF1 in the circulation andfacilitating binding between IGF1 and its receptor. The up regulation ofIGFBP5 and IGF2BP3 in the treated rat eyes demonstrated that activationof IGF pathway is one of the protection mechanisms induced byumbilicus-derived cells against cell death.

Another molecule that umbilicus-derived cells impacted was TXNL1. TXNL1is thioredoxin-related protein, a member of the thioredoxin pathway,which regulates oxygen stress and protects cells against oxygenstress-induced cell death. Oxygen stress stimulation such as hydrogenperoxide treatment induced apoptosis in cultured RPE cells. Thus, theincreased activity of thioredoxin pathway potentially could release theoxygen stress and protect RPE. Indeed, this pathway has been shown upregulated in non-apoptotic RPE cell line compared to apoptosis RPE. Atday 7-post cell transplantation, the up-regulation of cell proliferationand protection against cell death, either by increasing the activity ofIGF signaling pathway or by releasing oxygen stress, paves the way forpreserved photoreceptors in the outer nuclear layer and preserved visualfunction in RCS rats.

The predominant anti-apoptosis pathways at day 7 continued to be thedominant signaling network at day 30 as shown in Table 27-2. Theplatelet-derived growth factor C (PDGFC) is unregulated at day 30 in thecell-treated eyes. PDGFC is a member of PDGF family. Similar to IGF1,PDGF family members also demonstrate a broad biological functions inmany cell types, including proliferation and survival.

Down-stream to the PDGF receptor, SH2B was up regulated in the treatedeyes. SH2B is a cytoplasmic adaptor protein that connects the growthfactor receptor to the kinase cascade. The up regulation of both thegrowth factor PDGFC and the adaptor protein SH2B demonstrated the impactby umbilicus-derived cells on this growth factor pathway. Thecontinuation of the growth factor activation by the initial treatmentdemonstrated the long-lasting trophic effect from umbilicus-derivedcells in the treated eyes and correlated with the retention ofumbilicus-derived cells in the eyes.

Pathway analysis from Day 60 data revealed a predominantphototransduction pathway in the treated eyes (Table 27-3). Thephototransduction pathway is shown in FIG. 6. Briefly, light activationcauses a graded change in cell surface membrane potential. Thetransmission of the pulses is mediated by opening or closing of ionchannels, which are regulated by cyclic guanosine monophosphate (cGMP).The series of biochemical changes that ultimately leads to a reductionin cGMP levels begins when a photon is absorbed by the photo pigment inthe receptor disks, which contain proteins called opsins. One of opsinsin rod cells that mediate the molecular events post light perception isrhodopsin (RHO). When the retinal moiety in the rhodopsin moleculeabsorbs a photon, its configuration changes. This change then triggers aseries of alterations in the protein component of the molecule, andleads to the activation of an intracellular messenger called transducin,which activates a phosphodiesterase that reduces the concentration cGMPand leads to channel closure at cell surface membrane (FIG. 6).

Molecules involved in the phototransduction pathway such as CNGA1,GNAT1, GNB1, RHO, and PDE6B were up regulated. These genes distributedin the entire photoreceptor transduction network. Cyclicnucleotide-gated (CNG) ion channel subunit CNGA1 expression is increasedmore than 6 fold in the treated eyes and located at photoreceptor outersegment in the transduction pathway to eventually mediate the membranepotential pulses in photoreceptors as shown in FIG. 6. RHO is one of theproteins in opsins, which is a complex of molecules to receive andprocess photons from the light. The up-regulation of RHO suggestspreserved light perception function in rod cells. GNAT1 and GNBlarealpha and beta units in transducin that couple RHO andcGMP-phoshodiesterase during visual impulses. The expression of othervisual function regulatory molecules, PDE6B and ROCK1, which directlyregulate cGMP were also up regulated. Interestingly, the predominatingpathway transition from anti-apoptotic pathway at day 7 and 30 tophototransduction pathway at day 60, suggests a direct impact ofumbilicus-derived cell treatment on the protection of photoreceptors.

Trophic Factors Secreted by Umbilicus-Derived Cells:

From the microarray results, it is evident that umbilicus-derived cellsmay play a role in promoting proliferation and reducing apoptosis atearly stage. Additionally, umbilicus-derived cells may play a role inpreventing photoreceptor cell death, and maintaining phototransductionat later stage. To determine the cytokines secreted by umbilicus-derivedcells that can impact on proliferation and protection, we looked intothe trophic factors that are secreted by umbilicus-derived cells invitro.

From the growth factor analysis, IL-8, IL-6, HGF, and IGF-1 are highlysecreted by umbilicus-derived cells in vitro (Table 27-4 and Table27-5). The pathway analysis described above has demonstrated theinvolvement of IGF pathway in the umbilicus-derived cell treated eyes.Other cytokines that are secreted by umbilicus-derived cells in vitroinclude basic FGF, BDNF, CNTF, NT3 as shown in Table 27-4. The trophicfactor profiles correlated with the findings from microarray.

Example 28 Conditioned Medium from Umbilicus-Derived Cells ProtectedARPE-19 from H₂O₂-Induced Apoptosis

Apoptosis may be involved in the development of retinal degenerationdiseases such as RP and AMD. Apoptosis in photoreceptors is derived frommultiple pathophysiologies, including defects in the process ofphagocytosis and oxygen stress. Studies from animal models have shown adeleterious effect in photoreceptors and RPE from oxidative damages. Theoxidative damage can form a cycle of cell death initiated from the rodundergoing apoptosis. The apoptotic rod cells created environment withincreased blood flow and higher oxygen in the retina, which lead todeterioration of RPE, rods, and cones, which is the major cell type forvision.

In the previous example, a gene profiling study (Microarray) revealedanti-apoptosis as a key signaling pathway at early stage post celltransplantation and preservation of phototransduction at later stage.Follow-up trophic factor assessment in the umbilicus-derived cellconditioned media has confirmed the microarray results and identifiedseveral cytokines as anti-apoptotic agents based on literatures. In thisstudy, we intend to evaluate if umbilicus-derived cell conditioned mediacan protect RPE cell line from apoptosis induced by hydrogen peroxide(H₂O₂) and to develop a cell-based apoptosis assay.

Materials and Methods Umbilicus-Derived Cell Culture for ConditionedMedia Collection:

Frozen vials of previously expanded umbilicus-derived cells (populationdoubling 20) were plated at 5,000 cells/cm² on T75 flasks in GrowthMedium as described above. The flasks were incubated in a 37° C.incubator for 24 hours. The supernatant of the culture was collectedafter 24 hour culture, and saved frozen at −80° C. or applied to ARPEcultures immediately.

ARPE-19 Culture:

Frozen vials of previously expanded ARPE-19 cells (CRL-2502, AmericanType Culture Collection, Manassas, Va.) were seeded onto 24-well platesin Growth Medium at 40,000 cells/well or in 96-well plate at 5000/wellfor 24 hours. Cells were washed with PBS and treated with mediacontaining H₂O₂.

H₂O₂ Treatment:

H₂O₂ was prepared by adding H₂O₂ at different concentrations (0, 0.125mM, 0.25 mM, 0.5 mM, 1.0 mM, 1.2 mM) to the growth media (GM) orconditioned media (CM) from umbilicus-derived cell culture. ARPE-19 werecultured in H₂O₂ containing media for 0, 0.5 hr, 1 hr, 2 hr, 3 hr, thenthe H₂O₂ media was removed and cells were used for further apoptosisassays.

For Annexin V assay, ARPE-19 cells were treated with wither growth mediaor conditioned media for 24 hours before assessment of apoptosis.

Colometric Apoptosis Assay:

The apoptosis assay was performed according to the instruction in theCell Death Detection ELISAplus kit from Roche (Cat #11 774 425 001).Briefly, the cells still attached to the plate was lysed and incubatedwith an antibody against histone to pull histone from the whole lysates.Another antibody, linked with HRP, recognizing the DNA fragments will beused to identify the DNA components within the histone section. Theapoptotic complex of histone and DNA fragments will be recognized by HRPsubstrates.

Annexin V Apoptosis Assay:

After a 24 hour incubation period, the culture medium was removed andthe plate was washed with PBS. Trypsin (Gibco) was added to dislodge thecells from the individual wells. The culture medium from each well andthe trypsinized cells from each respective well were combined andcentrifuged at 250×g for 5 minutes and the pellet was resuspended in 404cold 1× Nexin buffer. Five microliters Annexin V-PE and 54 Nexin 7-AADwere added to each sample and the mixture was vortexed and incubatedshielded from light at 4° C. for 20 minutes. Following incubation, 450μL of cold 1× Nexin buffer was added to each sample and the suspensionwas vortexed. The stained samples were loaded onto the Guava PersonalCytometer and were acquired and analyzed according to the Guava PersonalCytometer User's Guide.

Results

Time- and Dose-Dependent Apoptosis Induction by H₂O₂ in ARPE-19 Cells:

H₂O₂ induced apoptosis in a time- and dose-dependent manner. Apoptosismeasured by DNA fragments associated with histones was determined usingELISA. When ARPE-19 cells were treated with a series of concentrationsof H₂O₂ from 0.25 mM to 1.2 mM, DNA fragmentation was at 1-fold ofnegative control (0 mM) at 0.5 mM and was increased to 2.6-fold and 3.3fold at 1 mM and 1.2 mM respectively, shown in FIG. 7.

In a separate experiment, apoptosis was measured by measuring thepercentage of Annexin V(+) cells in the population. One of the earlyevents during apoptosis process is the exposure of a phospholipid-likephosphatidylserine (PS) to the cell surface membrane. Annexin V is amolecule that can bind to PS on the cell surface. Using this feature,apoptotic cells can be detected by staining the cell surface withAnnexin V. The percentage of cells in the population that are either inthe early stages of apoptosis or the later stages of apoptosis wasdetermined by measuring changes in cell membrane permeability to the DNAdye 7-AAD. At the early stages of apoptosis, when the cell membrane isstill intact, 7-AAD is unable to penetrate the cell membrane and will benegative. Therefore early apoptotic cells are typified as being AnnexinV(+) and 7-ADD(−). At later stages of apoptosis, when the cell membraneintegrity fails, and can be penetrated by 7-ADD, the apoptotic cells aredemonstrated as Annexin V(+) and 7-ADD (+).

Early apoptotic cells, as determined by the percentage of cells thatwere Annexin V(+) and 7-AAD(−) increased with the concentration of H₂O₂.At 0.125 mM H₂O₂, the percentage of cells that were Annexin V(+) and7-AAD(−) was 8%. This increased to 18% at 0.25 mM H₂O₂ and reached amaximum at 34% at 0.5 mM H₂O₂. Similarly, total apoptotic cells, asdetermined by measuring the total Annexin V(+) cells in the populationwas 16% at 0.125 mM H₂O₂, that increased to 27% at 0.25 mM H₂O₂. Theerect of H₂O₂ on the total number of apoptotic cells was maximal at 0.5mM H₂O₂ (FIG. 8). When ARPE-19 was treated at 1.2 mM but with differentincubation time, a time course effect was evident. DNA fragmentationreached its plateau by 1 hr, about 3.3-fold versus 0 hr incubation, andremained at this level to 3 hrs as shown in FIG. 9.

Umbilicus-Derived Cell Conditioned Media Reduced Apoptosis Both Dose-and Time-Dependently:

The DNA fragmentation induced by H₂O₂ was decreased when cells weretreated with conditioned media from umbilicus-derived cells. DNAfragment levels at different doses and different incubation time inconditioned media were maintained at the similar level as the negativecontrol from 0.25 mM to 1.2 mM (0.5-fold to 1.5-fold vs. control), shownin FIGS. 7 and 9.

Similar results were seen using the Annexin V assay. Early apoptosisevents and total apoptotic cells were reduced in umbilicus-derived cellconditioned media-treated ARPE-19 cells at 0.25 mM, 0.5 mM, and 1.0 mMas shown in FIG. 8.

These results confirmed the findings in literature that H₂O₂ is a potentstimulator of apoptosis for RPE cells. Most importantly, conditionedmedia from umbilicus-derived cells reduced apoptosis measured by bothDNA fragmentation and Annexin V positivity. The dose- and time-dependentinhibition of apoptosis by the conditioned media strongly suggests thatcytokines secreted by umbilicus-derived cells can rescue RPE cells fromoxygen stress-induced apoptosis.

Example 29 Preservation of Multiple Cell Types in the Retina of aPreclinical Model of Retinitis Pigmentosa

The Royal College of Surgeons (RCS) rat is a preclinical model ofretinitis pigmentosa in which there is progressive degeneration of therod and cone photoreceptors resulting from a specific defect in theretinal pigmented epithelial cells (RPE). In this model, a mutation inthe allele of the gene for the receptor tyrosine kinase Mertk results inan inability of the RPE to phagocytose shed rod outer segments (ROS).This defect results in apoptotic photoreceptor cell death, beginningaround post-natal day 20 (P20), and by P60 the outer nuclear layer (ONL)of these animals, which contains the photoreceptor cell nuclei, is only1-2 layers in thickness.

Umbilicus-derived cells are an allogeneic cell type with potential forcell therapy applications. Umbilicus-derived cells are obtained from anethical cell source and can be readily expanded to yield at least 1×10¹⁷cells from a single donor without karyotypic or phenotypic changes. Asingle dose of 20,000 umbilicus-derived cells into the subretinal spaceof RCS rats can preserve visual function as assessed by the use ofelectroretinogram, optomoter and luminance threshold testing (See, forexample, Lund et al. 2007). Umbilicus-derived cells have alsodemonstrated superior efficacy in preserving visual function compared totwo other allogeneic expandable tissue-derived cell types;placental-derived cells and human bone marrow mesenchymal stem cells(hMSC) (Lund et al. 2007). Furthermore, anatomical analysis demonstratedthat umbilicus-derived cells at 80 days post cell injection can preserve4-5 layers of cell nuclei in the outer nuclear layer (ONL) of thesubretinal space compared to sham or untreated controls which at thesame time point only have 1 layer of cells retained in the ONL.

While histology has been used to visualize the effects ofumbilicus-derived cell therapy on the retina, it has been mostlyanecdotal in nature and not quantitative. The goal of this study was touse quantitative morphometry to characterize the preservation of variouscell types in the retina important in rod photoreceptor connectivity andsignaling following subretinal umbilicus-derived cell administration inRCS rats.

Materials and Methods Animals:

Experiments were performed on male and female pigmented dystrophic RCSrats (rdy−/p−), which were individually housed with a 12-hour light/darkcycle at the Moran Eye Center (University of Utah, Salt Lake City,Utah). All procedures were approved and monitored by the University ofUtah Institutional Animal Care and Use Committee and have been conductedin accordance with the Policies on the Use of Animals and Humans inNeuroscience Research, revised and approved by the Society forNeuroscience in January 1995. The rats weighed approximately 30-40 gramsat age of dosing (post-natal day 21). Food and water were available adlibitum.

Preparation of Donor Cells:

Human umbilical cords were obtained with donor consent following livebirths from the National Disease Research Interchange (Philadelphia,Pa.) and umbilicus-derived cells were isolated, tested and cryopreservedas described in Examples 1-5 above. Prior to injection,umbilicus-derived cells were thawed rapidly in a 37° C. water bath,washed twice in sterile PBS and resuspended at a final concentration of1×10⁴ cells/μL.

Injection Procedure:

On Day 0 (post-natal Day 21) the rats received a subretinal injection of20K umbilicus-derived cells in a volume of 2 μL. RCS rats wereanesthetized with an intraperitoneal injection of xylazine-ketamine (1mg/kg of the following mixture: 2.5 ml xylazine at 20 mg/ml, 5 mlketamine at 100 mg/ml, and 0.5 ml distilled water). 20Kumbilicus-derived cells were injected through a fine glass pipette(internal diameter 75-150 μm) into the eye through a small scleralincision. The incision was closed with a small suture after injectionwas completed. All animals received daily dexamethasone injections (1.6mg/kg, i.p.) for 2 weeks post-injection, and received cyclosporine-A(Bedford Labs, Bedford Mass.) administered in the drinking water (210mg/L; resulting blood concentration: 250-300 μg/L) from 1-2 days priorto cell injection until euthanasia. Rats were sacrificed at thefollowing time points after surgery: 1, 7, 14, 30 and 60 days.Euthanasia was performed by anesthesia with aketamine:xylazine:acepromazine mixture, followed by exsanguination.

Collection of Eyes and Extraction of RNA for RT-PCR:

Following euthanasia, the eyes were extracted and placed in RNAlatersolution (Ambion, Austin, Tex.). All eyes were kept at 4° C. in RNAlatersolution for up to 3 days, then stored at −80° C. until processing. Eyeswere placed in Lysing Matrix tubes (Qbiogene, Carlsbad, Calif.)containing 1.4 ml Trizol (Invitrogen, Carlsbad, Calif.) and centrifugedin a Fast Prep FP120 homogenizer (Qbiogene, Carlsbad, Calif.) at speed#6 for 45 sec. The supernatant was transferred to a 5 ml round bottomtube. Trizol (2-4 ml) was added to the supernatant and samples were thencentrifuged at 9000 rpm for 10 min. Chloroform (2-3 ml) was added to thesupernatant and incubated at room temperature then centrifuged at 9000rpm for 10 min at room temperature. Isopropanol (3-4 ml) was added tothe supernatant to precipitate total RNA. The RNA pellet was obtained bycentrifuging at 9000 rpm for 10 min. The pellet was washed with 70%ethanol and air-dried at room temperature, then resuspended in 100 μlRNase-free water and stored at −80° C. The RNA quality and quantity wasanalyzed using the Experion automated electrophoresis system (Bio-RadLaboratories, Hercules, Calif.).

2-Step RT-PCR:

First-strand cDNA synthesis was performed using the First-Strand cDNASynthesis Kit (Invitrogen, Carlsbad, Calif.). Briefly, 10 μg total RNAwas mixed with 2 μl oligo(dT)20 (50 μM) and 2 μl dNTP (10 mM), incubatedat 65° C. for 5 min, then added 4 μl 10× reaction buffer, 8 μl 25 mMMgCl2, 4 μl 0.1M DTT, 2 μl RNaseOUT and 2 μl SUPERSCRIPT II RT enzyme,incubated at 50° C. for 50 min, and 85° C. for 5 min. Template RNA wasremoved by addition of 1 μl RNase H for 20 min at 37° C. Real-time PCRwas performed on the 7900HT Fast Real Time PCR System (AppliedBiosystems, Foster City, Calif.) using human β-2 microglobulin (hβ2M)TAQMAN Gene Expression Assay with the TAQMAN Fast Universal PCR MasterMix (Applied Biosystems, Foster City, Calif.). All samples were analyzedin triplicate. RNA standards were run alongside samples for quantitationof the number of cells present in injected eyes. To generate RNAstandards, eyes from Sprague-Dawley rats were collected as describedabove. The eyes were injected with 160, 800, 4K, 10K, and 20Kumbilicus-derived cells (n=3). RNA extraction and two-step real-timeRT-PCR was performed as described above. The threshold cycle value wasobtained and analyzed against the injected cell numbers.

Collection and Processing of Eyes Fro Histology:

Following euthanasia, the rat was flushed with phosphate buffered salineunder low pressure via the aorta. The eyes were carefully removed, andsurrounding tissue from the eye trimmed. Both the right and the left eyefrom each animal were collected and immersed in Pen-Fix fixative(Richard Allan Scientific) for 24-48 h. After fixation, the eyes wereprocessed for histology including dehydration, clearing in xylene andinfiltration with paraffin. Each eye was specifically oriented duringembedding in paraffin: the superior pole of the eye was embedded down inthe paraffin block and the suture marking the injection site rotated ina clockwise manner until it was approximately in the 3 o'clock positionon the globe. This orientation was maintained during embedding. Thisresulted in the injection site residing approximately in the centralplane of the eye for the majority of the eyes processed. For injectedeyes, collection of 5 μm sections began after facing into the blockuntil the suture marking the injection site is identified on astereomicroscope (FIG. 10). If no suture was identified, the eye wasexcluded from further analysis, as was its uninjected counterpart (lefteye from same animal). If the suture was identified after sectioningthrough the central plane of the eye and it was determined that itresided near the pole of the eye, it was excluded from further analysis,as was its uninjected counterpart. For uninjected eyes, sectioning beganafter the area/depth of interest is identified in the injected (right)eye. The corresponding uninjected (left) eye from the same animal wassectioned to a similar depth, collecting sections from approximately thesame area of the eye.

Antibodies:

The following antibodies were used for immunohistochemistry:rabbit-anti-Rhodopsin (Chemicon), rabbit anti-Calretinin (Chemicon),rabbit anti-Recoverin (Chemicon), mouse-anti-human nuclear matrixantigen (NuMA, Calbiochem). Antibodies for secondary detection werebiotinylated anti-rabbit (Chemicon), and biotinylated goat-anti-mouse(Jackson Immunoresearch).

Immunocytochemistry:

Sections for IHC staining were incubated for 1 hr at 59° C. followed bydeparaffinization through a series of changes in xylene, 100% alcohol,95% alcohol, and water on a TISSUE TEK® DRS™ 2000 Slide Stainer (Sakura,Torrance, Calif.). Slides were rinsed with tap water for approximately 5minutes. All immunohistochemical staining was performed on the i6000™Automated Staining System (BioGenex, San Ramon, Calif.). Antigenretrieval was performed when necessary using a Decloaking Chamber(BioCare Medical, Concord, Calif.) and Reveal HIER Solution (BioCareMedical) or microwaved using Antigen Retrieval Citra Solution(BioGenex). Endogenous activity of peroxidase and antigenic sites wereblocked and normal goat serum (BioGenex) reduced background staining dueto non-specific binding of the primary or secondary antibody. Sectionswere incubated with primary antibody at room temperature, detection ofthe bound primary is achieved by the addition of a biotinylatedsecondary antibody+peroxidase-conjugated streptavidin (BioGenex) andperoxidase activity was made visible with diaminobenzidine (DAB)(BioGenex). Counterstaining with Mayer's Hematoxylin (BioGenex) for 1min.

Image Acquisition and Analysis:

A Nikon Eclipse E800 (Nikon Corporation, Tokyo, Japan) microscope wasequipped with an Evolution™ MP 5.0 RTV color camera (Media Cybernetics,Inc. Silver Spring, Md.), interfaced with an IBM computer (InternationalBusiness Machines Corporation, Armonk, N.Y.) running Windows 2000(Microsoft Corporation, Redmond, Wash.). Images were captured andanalyzed using Image-Pro Plus software version 5.1 (Media Cybernetics,Inc. Silver Spring, Md.). Microsoft Excel 2000 (Microsoft Corporation,Redmond, Wash.) and GraphPad Prism version 4.03 (GraphPad Software, Inc.San Diego, Calif.) were used to interpret, analyze and graph the rawdata. SigmaStat Statistical Software version 2.03 (SPSS, Inc. Chicago,Ill.) was used to perform statistical analysis on the collected data.Using the Auto-Pro tool within the Image-Pro Plus software, customwritten macros were used to perform the analysis consistently.

Morphometry of Day 60 Eyes:

Three umbilicus-derived cell injected and three control (uninjected)eyes from the day 60 group (4 animals) were imaged and analyzed withmorphometry. The images captured were 24-bit RGB images, 2560×1920pixels in size, with a resolution of 300×300 dots/inch. The images werecaptured with a 60× objective Nikon lens. No imaging or analysis wasperformed on areas of the retina that were torn, damaged, folded ormissing.

Measurement of Area of Outer Nuclear Layer (ONL) Per Length:

From each eye, three hematoxylin and eosin (H&E) stained sections, eachseparated by 20 μm in depth were imaged and analyzed for the ONLmeasurements. The areas where the images were collected were defined asregions 1 and 2. Region 1 is an area near the injection site and region2 is an area away from the injection site. Up to ten images (five fromeach region) were collected from each section. Using Image-Pro Plus, theONL was selected as the area of interest (AOI). The AOI was extractedand transformed into an 8-bit grayscale image, referred to as a mask.Area and length (to normalize the data) of the ONL were measured andused to calculate the area per length of the ONL for each image.

Measurement of Amount of Rhodopsin Immunostaining in the NeuroepithelialLayer:

One section per eye immunohistochemically stained for rhodopsin wasimaged and analyzed. The area where the images were collected wasdefined as region 1, an area near the injection site. Up to eight imageswere collected from each section. Using Image-Pro Plus, the saturationchannel of the color model HSI was extracted from the RGB image. Usingvarious acquired images with mixed saturation levels, a threshold wasset to the most intensely stained colors or the most dominant hues. Thisset threshold was used to analyze each image. The AOI was defined as theneuroepithelial layer in each image. The measurement calculated for eachimage was the percent area of the AOI that was within the set threshold.

Measurement of Area of the Calretinin Immunostaining Per Length:

One section per eye, immunohistochemically stained for calretinin wasimaged and analyzed. The area where the images were collected wasdefined as region 1, an area near the injection site. Up to eight imageswere collected from each section. Using Image-Pro Plus, three AOIs weredefined as: all recoverin-positive areas, the inner nuclear layer andthe outer ganglion cell layer (each analysis was performed separately)and were extracted from the image and transformed into an 8-bitgrayscale image, referred to as a mask. Area and length (to normalizethe data) of the calretinin staining were measured and used to calculatethe area per length of the calretinin staining for each image.

Measurement of Area of the Recoverin Immunostaining Per Length:

One section per eye, immunohistochemically stained for recoverin, wasimaged and analyzed. The area where the images were collected wasdefined as region 1, an area near the injection site. Up to eight imageswere collected from each section. Using Image-Pro Plus, three AOIs weredefined as: all recoverin-positive areas, the inner nuclear layer andthe outer nuclear layer (each analysis was performed separately). EachAOI was extracted from the image and transformed into an 8-bit grayscaleimage, referred to as a mask. Area and length (to normalize the data) ofthe recoverin staining within each AOI was measured and used tocalculate the area per length of the recoverin staining for each image.

Results Identification of Umbilicus-Derived Cells in Day 1 PostInjection:

Umbilicus-derived cell injected (right) eyes from day 1 post-injectionwere sectioned for the purposes of identifying injectedumbilicus-derived cells to confirm their placement in the eye. Usingthis method, injected human cells were positively identified usingimmunohistochemistry for human nuclear matrix antigen (NuMA, FIG. 12).

Umbilicus-Derived Cell Retention in the RCS Rat Eyes:

Umbilicus-derived cell retention in the RCS eyes was investigated usingRT-PCR for a human-specific antigen, β2 microglobulin ((32M) mRNA. TotalRNA from injected eyes was amplified using β2M specific primers. Valuesare converted to cell number using a standard curve generated usingtotal RNA isolated from eyes injected intravitreally with known numbersof umbilicus-derived cells. Despite variability between specimens, eyesat all time points had detectable levels of β2M mRNA. This indicatedthat the umbilicus-derived cells were retained in the RCS eye throughthe course of the study, although the cell number decline substantiallyby Day 60, with only approximately 10% of the injected cells detected(FIG. 13).

Morphometry of the Outer Nuclear Layer:

H&E-stained sections of both control (uninjected) and umbilicus-derivedcell-injected eyes from the following time points were examined: 7, 14,30 and 60 days post-injection (FIG. 14). The earliest time point inwhich ONL degeneration could be detected in control eyes was Day 30. Atthis time point, the effect of umbilicus-derived cell injection wassubtle. However, at Day 60, the effect of umbilicus-derived cellinjection was evident. In control eyes, the ONL had thinned to adiscontinuous layer 1-2 nuclei in thickness. In the eyes that hadreceived a subretinal injection of umbilicus-derived cells, the ONL wasapproximately 3-4 nuclei in thickness near the injection site region,looking comparable to the ONL in the Day 30 specimens. This suggested apreservation of the ONL by umbilicus-derived cells.

Eyes from day 60 post-injection were evaluated for the effect ofumbilicus-derived cells on outer nuclear layer (ONL) thickness, as ameasure of photoreceptor rescue. Images were taken at 60× magnificationfrom two regions of the retina, near and far from the injection site inH&E stained sections (see FIG. 11). Two eyes were excluded from analysisdue to poor morphology and loss of the majority of the retina,presumably caused by inadequate fixation. The ONL was visibly thicker inumbilicus-derived cell injected eyes compared with control (uninjected)eyes in both regions examined (FIG. 15A-D). Analysis of the images usingmorphometry supported this observation: the area per length occupied byONL nuclei was 7.5 times higher near the injection site, 2.6 timeshigher in areas far from the injection site in umbilicus-derived cellinjected eyes compared with control eyes, and 5 times higher overallwhen the results from both regions were combined, suggesting that theumbilicus-derived cells are preserving photoreceptors in the RCS rat(FIGS. 15E and F). This also shows that the effect is greatest local tothe injection site, however there is an overall effect ofumbilicus-derived cell injection.

Image Analysis of Rhodopsin Immunostaining:

Eyes from day 60 post-injection were evaluated for the effect ofumbilicus-derived cells on expression of rhodopsin usingimmunohistochemistry and image analysis. As with the ONL analysis, avisible difference could be seen in the level of rhodopsinimmunostaining in umbilicus-derived cell injected eyes compared withcontrol eyes (FIGS. 16A and B). Images at 60× magnification wereacquired from regions near the injection site of these eyes. Forquantitation of rhodopsin immunostaining, the saturation channel of thecolor model HSI was extracted from the RGB image. Saturation refers tothe dominance of hue in the color. A dominant hue is considered a purecolor and a less dominant hue is a lighter shade of a pure color, forinstance pink is red with a low degree of saturation or dominance. Thesaturation value, or amount of rhodopsin staining, translates to howdensely packed the rods and cones are within the neuroepithelial layer.A highly saturated area is most likely an area of tightly packed rodsand cones. Near the injection site region, the rhodopsin immunostainingwas 10 fold higher in umbilicus-derived cell injected eyes compared withcontrols (FIG. 16C), translating to more densely packed rod outersegments containing rhodopsin and indicating preservation of rodphotoreceptors.

Image Analysis of Calreticulin Immunostaining:

Eyes from day 60 post-injection were evaluated for the effect ofumbilicus-derived cells on expression of calretinin usingimmunohistochemistry and image analysis. Unlike the ONL and rhodopsinimmunostaining, there was not a marked difference visible in the imagesof calretinin immunostaining in control and umbilicus-derived cellinjected eyes, however upon quantitation there was a small butstatistically significant difference between the two groups. Images at60× magnification were acquired from regions near the injection site ofthese eyes (FIGS. 17A and B). Quantitation of calretinin immunostainingwas performed in the following layers: inner nuclear layer (INL), innerplexiform layer (IPL) and retinal ganglion cell layer (GCL) together,and the INL and GCL individually. Near the injection site region,overall calretinin immunostaining in all 3 layers was 1.2 fold higher inumbilicus-derived cell injected eyes compared with controls. In the GCL,there was no difference between control eyes and umbilicus-derivedcell-injected eyes, however there was a 1.5 fold increase in calretininimmunostaining in the INL of umbilicus-derived cell-injected eyescompared to controls (FIGS. 17C and D), indicating preservation ofcalretinin-expressing cells in the INL.

Image Analysis of Recoverin Immunostaining:

Eyes from day 60 post-injection were evaluated for the effect ofumbilicus-derived cells on expression of recoverin usingimmunohistochemistry and image analysis. Images at 60× magnificationwere acquired from regions near the injection site of these eyes (FIGS.18A and B). Quantitation of recoverin immunostaining was performed onthe inner nuclear layer (INL) and the outer nuclear layer (ONL). Nearthe injection site region, the recoverin immunostaining was 2 foldhigher in the INL and 7.9 fold higher in the ONL of theumbilicus-derived cell injected eyes compared with controls (FIGS. 18Cand D), indicating preservation of recoverin-expressing cells.

DISCUSSION

Umbilicus-derived cell injection into the subretinal space of RCS ratshas been demonstrated to sustain visual function for several monthsafter injection into the subretinal space of RCS rats (Lund et al.2007). It has also been shown to preserve photoreceptor nuclei in theONL, however this was not done quantitatively. The goal of this studywas to quantitate the effects of umbilicus-derived cell injection on theretina using morphometry. To analyze both ONL thickness as well asquantitation of various retinal cell types using immunohistochemistry,it was crucial to orient the eyes identically in the paraffin block.Additionally, it was necessary to obtain sections near themidline/central plane of the eye, so that the retinal layers to beanalyzed would not appear artificially thick due to taking a slicethrough the curvature of the eye. Great care was taken to attain both ofthese requirements. By using such a careful approach in maintainingorientation of the eyes during processing, embedding and sectioning, thelocation of the injection site region was known in every eye.

Only the 60 days post-injection time point in this study was selectedfor morphometric analysis, as this time point clearly showed an effectof the injected hUTC on ONL thickness in H&E stained sections. Theeffect of hUTC injection on the ONL was evaluated morphometrically. TheONL contains the cell bodies of the photoreceptor cells, and isordinarily packed with nuclei arranged in 10-12 rows. In retinaldegenerative diseases such as retinitis pigmentosa, photoreceptor celldeath results in a thinning of the ONL to approximately 1 layer ofnuclei in thickness. There was a visible difference in ONL thicknessbetween control (uninjected) and umbilicus-derived cell injected eyes 60days post-injection, and this difference was supported morphometrically.Areas both near and far from the injection site were significantlyincreased in umbilicus-derived cell injected eyes, suggestingumbilicus-derived cell injected animals preserved greater numbers ofnucleated cells in the ONL compared with control animals, thusphotoreceptor rescue in these animals. This effect was greatest in theregion near the injection site; therefore this region was selected foranalysis of rhodopsin, calretinin and recoverin immunostaining.

Rhodopsin immunostaining in the neuroepithelial layer was also visiblyincreased in the eyes that received injection of umbilicus-derivedcells. The neuroepithelial layer contains the rods and cones' outersegments, which are specialized processes of the photoreceptor neuronslocated in the ONL. Rod outer segments (ROS) convert and amplify thelight signal. Mammalian ROS are packed with stack of 1000-2000 flatteneddisks that are formed by invaginations of the plasma membrane. The discsof the ROS are responsible for trapping photons and express a high levelof rhodopsin, also known as visual purple. Rhodopsin is also expressedat a lower level on the plasma membrane. ROS are renewed and shed by therod photoreceptors daily, and the shed ROS are phagocytosed and recycledby the RPE. Highly saturated rhodopsin immunostaining was significantlyincreased in umbilicus-derived cell injected animals compared withcontrols, indicating the presence of greater numbers of tightly packedrhodopsin-expressing ROS in the neuroepithelial layer of the retina.This further suggests the rescue/preservation of rod photoreceptorscontaining functional outer segments and is consistent with the resultthat there is photoreceptor rescue in the ONL.

Immunostaining for the calcium-binding protein, calretinin, was alsoevaluated. Calretinin is expressed in neurons of the central nervoussystem, but its precise function has not yet been elucidated. In therat, calretinin expression has been demonstrated in amacrine cellslocated in the INL, retinal ganglion cells in the GCL, and labels 3bands in the IPL, the site for synaptic junctions between the INL andGCL. All three layers staining positively for calretinin werequantitated. While there was not an impressive difference that could bedetected visually in the calretinin staining between control andumbilicus-derived cell injected eyes, there was a small butstatistically significant increase found morphometrically. This 1.2 foldincrease in overall calretinin immunostaining suggested a preservationof calretinin-expressing cells in the retina. Quantitation of calretininimmunostaining was subsequently performed on the INL and GCLindividually to determine if this effect was specific to a particularlayer of the retina. There was no difference in the calretinin stainingin the GCL, however there was a 1.5 fold increase in staining in theINL, suggesting a preservation of calretinin-expressing cells in theinner nuclear layer, possibly amacrine cells.

Recoverin immunostaining was performed on the day 60 specimens.Recoverin is a calcium-binding protein localized to photoreceptor cellbodies in the outer nuclear layer, as well as midget cone bipolar cellsin the inner nuclear layer. Recoverin immunostaining was visiblyincreased in umbilicus-derived cell-injected eyes compared withcontrols, suggesting a preservation of one or more cell types.Morphometry was performed on recoverin staining in the INL and ONL.There was a statistically significant increase in recoverinimmunostaining in umbilicus-derived cell injected eyes compared withcontrols in both layers evaluated, suggesting a preservation ofphotoreceptors in the ONL and of recoverin-expressing cells in the INL,possibly cone bipolar cells.

Taken together, the increase in ONL thickness, rhodopsin, calretinin,and recoverin immunostaining in umbilicus-derived cell-injected eyescompared with controls suggest preservation in the structure of theretina through rescue of photoreceptors, functional rod outer segmentsand possibly bipolar neurons responsible for relaying information fromthe retina to the brain. These results provide support for thefunctional findings that umbilicus-derived cell subretinal injectionpreserves vision in dystrophic RCS rats. The mechanism of action ofumbilicus-derived cells remains to be elucidated. There has been noevidence of differentiation of umbilicus-derived cells intophotoreceptors or other retinal cell types. In fact, only a smallfraction (less than 10%) of injected umbilicus-derived cells aredetectable by quantitative RT-PCR for a human-specific gene 60 dayspost-injection. If the cells do not survive over the course of time inwhich there is a therapeutic effect, perhaps the mechanism is that thecells secrete a neurotrophic factor which slows the apoptotic wave inthe photoreceptor population, thereby allowing for preservation of notonly photoreceptors, but their supporting cells as well.Umbilicus-derived cells are currently being characterized in vitro toaddress secretory factors and mechanistic studies are in progress.

Publications cited throughout this document are hereby incorporated byreference in their entirety. Although the various aspects of theinvention have been illustrated above by reference to examples andpreferred embodiments, it will be appreciated that the scope of theinvention is defined not by the foregoing description but by thefollowing claims properly construed under principles of patent law.

TABLE 1-1 Isolation of cells from umbilical cord tissue using varyingenzyme combinations Cells Cell Enzyme Digest Isolated ExpansionCollagenase X X Dispase  + (>10 h) + Hyaluronidase X XCollagenase:Dispase ++ (<3 h) ++ Collagenase:Hyaluronidase ++ (<3 h) +Dispase:Hyaluronidase  + (>10 h) + Collagenase:Dispase:Hyaluronidase +++(<3 h)  +++ Key: + = good, ++ = very good, +++ = excellent, X = nosuccess under conditions tested

TABLE 1-2 Isolation and culture expansion of postpartum cells undervarying conditions: Growth Condition Medium 15% FBS BME Gelatin 20% O2Factors 1 DMEM-Lg Y Y Y Y N 2 DMEM-Lg Y Y Y N (5%) N 3 DMEM-Lg Y Y N Y N4 DMEM-Lg Y Y N N (5%) N 5 DMEM-Lg N (2%) Y N (Laminin) Y EGF/FGF (20ng/mL) 6 DMEM-Lg N (2%) Y N (Laminin) N (5%) EGF/FGF (20 ng/mL) 7DMEM-Lg N (2%) Y N (Fibrone) Y PDGF/VEGF 8 DMEM-Lg N (2%) Y N (Fibrone)N (5%) PDGF/VEGF 9 DMEM-Lg Y N Y Y N 10 DMEM-Lg Y N Y N (5%) N 11DMEM-Lg Y N N Y N 12 DMEM-Lg Y N N N (5%) N 13 DMEM-Lg N (2%) N N(Laminin) Y EGF/FGF (20 ng/mL) 14 DMEM-Lg N (2%) N N (Laminin) N (5%)EGF/FGF (20 ng/mL) 15 DMEM-Lg N (2%) N N (Fibrone) Y PDGF/VEGF 16DMEM-Lg N (2%) N N (Fibrone) N (5%) PDGF/VEGF

TABLE 2-1 Growth characteristics for different cell populations grown tosenescence Total Population Total Cell Cell Type Senescence DoublingsYield MSC 24 d 8 4.72 E7 Adipose 57 d 24 4.5 E12 Fibroblasts 53 d 262.82 E13 Umbilicus 65 d 42 6.15 E17 Placenta 80 d 46 2.49 E19

TABLE 2-2 Growth characteristics for different cell populations usinglow density growth expansion from passage 10 till senescence TotalPopulation Total Cell Cell Type Senescence Doublings Yield Fibroblast(P10) 80 d 43.68 2.59 E11 Umbilicus (P10) 80 d 53.6 1.25 E14 Placenta(P10) 60 d 32.96 6.09 E12

TABLE 3-1 Culture Media Added fetal bovine serum % Culture MediumSupplier (v/v) DMEM low glucose Gibco Carlsbad CA 0, 2 10 DMEM highglucose Gibco 0, 2 10 RPMI 1640 Mediatech, Inc. 0, 2 10 Herndon, VA Cellgro-free (Serum-free, Mediatech, Inc. — Protein-free Ham's F10Mediatech, Inc. 0, 2 10 MSCGM (complete with Cambrex, 0, 2 10 serum)Walkersville, MD Complete-serum free Mediatech, Inc. — w/albumin GrowthMedium NA — Ham's F12 Mediatech, Inc. 0, 2 10 Iscove's Mediatech, Inc.0, 2 10 Basal Medium Eagle's Mediatech, Inc. DMEM/F12 (1:1) Mediatech,Inc. 0, 2 10

TABLE 6-1 Results of PPDC karyotype analysis Metaphase Metaphase Numberof Tissue passage cells counted cells analyzed karyotypes ISCN KaryotypePlacenta 22 20 5 2 46, XX Umbilical 23 20 5 2 46, XX Umbilical 6 20 5 246, XY Placenta 2 20 5 2 46, XX Umbilical 3 20 5 2 46, XX Placenta-N 020 5 2 46, XY Placenta-V 0 20 5 2 46, XY Placenta-M 0 21 5 4 46,XY[18]/46, XX[3] Placenta-M 4 20 5 2 46, XX Placenta-N 9 25 5 4 46,XY[5]/46, XX[20] Placenta-N C1 1 20 5 2 46, XY Placenta-N C3 1 20 6 446, XY[2]/46, XX[18] Placenta-N C4 1 20 5 2 46, XY Placenta-N C15 1 20 52 46, XY Placenta-N C20 1 20 5 2 46, XY Key: N—Neonatal aspect;V—villous region; M—maternal aspect; C—clone

TABLE 7.1 Antibody Manufacture Catalog Number CD10 BD Pharmingen (SanDiego, 555375 CA) CD13 BD Pharmingen 555394 CD31 BD Pharmingen 555446CD34 BD Pharmingen 555821 CD44 BD Pharmingen 555478 CD45RA BD Pharmingen555489 CD73 BD Pharmingen 550257 CD90 BD Pharmingen 555596 CD117 BDPharmingen 340529 CD141 BD Pharmingen 559781 PDGFr-alpha BD Pharmingen556002 HLA-A, B, C BD Pharmingen 555553 HLA-DR, DP, DQ BD Pharmingen555558 IgG-FITC Sigma (St. Louis, MO) F-6522 IgG-PE Sigma P-4685

TABLE 9-1 Cells analyzed by the microarray study. Cell lines are listedby identification code along with passage at time of analysis, cellgrowth substrate and growth medium. Sub- Cell Population Passage strateMedium Umbilicus (022803) 2 Gelatin DMEM, 15% FBS, 2-ME Umbilicus(042103) 3 Gelatin DMEM, 15% FBS, 2-ME Umbilicus (071003) 4 GelatinDMEM, 15% FBS, 2-ME Placenta (042203) 12 Gelatin DMEM, 15% FBS, 2-MEPlacenta (042903) 4 Gelatin DMEM, 15% FBS, 2-ME Placenta (071003) 3Gelatin DMEM, 15% FBS, 2-ME ICBM (070203) (5% 02) 3 Plastic MEM, 10% FBSICBM (062703) (std. O2) 5 Plastic MEM, 10% FBS ICBM (062703) (5% 02) 5Plastic MEM, 10% FBS hMSC (Lot 2F1655) 3 Plastic MSCGM hMSC (Lot 2F1656)3 Plastic MSCGM hMSC (Lot 2F 1657) 3 Plastic MSCGM hFibroblast (9F0844)9 Plastic DMEM-F12, 10% FBS hFibroblast (CCD39SK) 4 Plastic DMEM-F12,10% FBS

TABLE 9-2 The Euclidean Distances for the Cell Pairs. Cell PairEuclidean Distance ICBM-hMSC 24.71 Placenta-umbilical 25.52ICBM-Fibroblast 36.44 ICBM-placenta 37.09 Fibroblast-MSC 39.63ICBM-Umbilical 40.15 Fibroblast-Umbilical 41.59 MSC-Placenta 42.84MSC-Umbilical 46.86 ICBM-placenta 48.41

TABLE 9-3 Genes shown to have specifically increased expression in theplacenta-derived cells as compared to other cell lines assayed GenesIncreased in Placenta-Derived Cells NCBI Accession Probe Set ID GeneName Number 209732_at C-type (calcium dependent,carbohydrate-recognition domain) AF070642 lectin, superfamily member 2(activation-induced) 206067_s_at Wilms tumor 1 NM_024426 207016_s_ataldehyde dehydrogenase 1 family, member A2 AB015228 206367_at reninNM_000537 210004_at oxidized low density lipoprotein (lectin-like)receptor 1 AF035776 214993_at Homo sapiens, clone IMAGE: 4179671, mRNA,partial cds AF070642 202178_at protein kinase C, zeta NM_002744209780_at hypothetical protein DKFZp564F013 AL136883 204135_atdownregulated in ovarian cancer 1 NM_014890 213542_at Homo sapiens mRNA;cDNA DKFZp547K1113 (from clone AI246730 DKFZp547K1113)

TABLE 9-4 Genes shown to have specifically increased expression in theumbilicus-derived cells as compared to other cell lines assayed GenesIncreased in Umbilicus-Derived Cells NCBI Accession Probe Set ID GeneName Number 202859_x_at interleukin 8 NM_000584 211506_s_at interleukin8 AF043337 210222_s_at reticulon 1 BC000314 204470_at chemokine (C-X-Cmotif) ligand 1 NM_001511 (melanoma growth stimulating activity206336_at chemokine (C-X-C motif) ligand 6 NM_002993 (granulocytechemotactic protein 2) 207850_at chemokine (C-X-C motif) ligand 3NM_002090 203485_at reticulon 1 NM_021136 202644_s_at tumor necrosisfactor, alpha-induced NM_006290 protein 3

TABLE 9-5 Genes shown to have decreased expression in umbilicus- andplacenta-derived cells as compared to other cell lines assayed GenesDecreased in Umbilicus- and Placenta-Derived Cells NCBI Accession ProbeSet ID Gene name Number 210135_s_at short stature homeobox 2 AF022654.1205824_at heat shock 27 kDa protein 2 NM_001541.1 209687_at chemokine(C-X-C motif) ligand 12 (stromal cell-derived factor U19495.1 1)203666_at chemokine (C-X-C motif) ligand 12 (stromal cell-derived factorNM_000609.1 1) 212670_at elastin (supravalvular aortic stenosis,Williams-Beuren AA479278 syndrome) 213381_at Homo sapiens mRNA; cDNADKFZp586M2022 (from clone N91149 DKFZp586M2022) 206201_s_at mesenchymehomeobox 2 (growth arrest-specific homeo box) NM_005924.1 205817_at sineoculis homeobox homolog 1 (Drosophila) NM_005982.1 209283_at crystallin,alpha B AF007162.1 212793_at dishevelled associated activator ofmorphogenesis 2 BF513244 213488_at DKFZP586B2420 protein AL050143.1209763_at similar to neuralin 1 AL049176 205200_at tetranectin(plasminogen binding protein) NM_003278.1 205743_nt src homology three(SH3) and cysteine rich donatia NM_003149.1 200921_s_at B-celltranslocation gene 1, anti-proliferative NM_001731.1 206932_atcholesterol 25-hydroxylase NM_003956.1 204198_s_at runt-relatedtranscription factor 3 AA541630 219747_at hypothetical protein FLJ23191NM_024574.1 204773_at interleukin 11 receptor, alpha NM_004512.1202465_at procollagen C-endopeptidase enhancer NM_002593.2 203706_s_atfrizzled homolog 7 (Drosophila) NM_003507.1 212736_at hypothetical geneBC008967 BE299456 214587_at collagen, type VIII, alpha 1 BE877796201645_at tenascin C (hexabrachion) NM_002160.1 210239_at iroquoishomeobox protein 5 U90304.1 203903_s_at Hephaestin NM_014799.1 205816_atintegrin, beta 8 NM_002214.1 203069_at synaptic vesicle glycoprotein 2NM_014849.1 213909_at Homo sapiens cDNA FLJ12280 fis, clone MAMMA1001744AU147799 206315_at cytokine receptor-like factor 1 NM_004750.1 204401_atpotassium intermediate/small conductance calcium-activated NM_002250.1channel, subfamily N, member 4 216331_at integrin, alpha 7 AK022548.1209663_s_at integrin, alpha 7 AF072132.1 213125_at DKFZP586L151 proteinAW007573 202133_at transcriptional co-activator with PDZ-binding motif(TAZ) AA081084 206511_s_at sine oculis homeobox homolog 2 (Drosophila)NM_016932.1 213435_at KIAA1034 protein AB028957.1 206115_at early growthresponse 3 NM_004430.1 213707_s_at distal-less homeo box 5 NM_005221.3218181_s_at hypothetical protein FLJ20373 NM_017792.1 209160_ataido-keto reductase family 1, member C3 (3-alpha AB018580.1hydroxysteroid dehydrogenase, type II) 213905_x_at Biglycan AA845258201261_x_at Biglycan BC002416.1 202132_at transcriptional co-activatorwith PDZ-binding motif (TAZ) AA081084 214701_s_at fibronectin 1AJ276395.1 213791_at Proenkephalin NM_006211.1 205422_s_at integrin,beta-like 1 (with EGF-like repeat domains) NM_004791.1 214927_at Homosapiens mRNA full length insert cDNA clone AL359052.1 EUROIMAGE 1968422206070_s_at EphA3 AF213459.1 212805_at KIAA0367 protein AB002365.1219789_at natriuretic peptide receptor C/guanylate cyclase C AI628360(atrionatriuretic peptide receptor C) 219054_at hypothetical proteinFLJ14054 NM_024563.1 213429_at Homo sapiens mRNA; cDNA DKFZp564B222(from clone AW025579 DKFZp564B222) 204929_s_at vesicle-associatedmembrane protein 5 (myobrevin) NM_006634.1 201843_s_at EGF-containingfibulin-like extracellular matrix protein 1 NM_004105.2 221478_atBCL2/adenovirus E1B 19 kDa interacting protein 3-like AL132665.1201792_at AE binding protein 1 NM_001129.2 204570_at cytochrome coxidase subunit VIIa polypeptide 1 (muscle) NM_001864.1 201621_atneuroblastoma, suppression of tumorigenicity 1 NM_005380.1 202718_atinsulin-like growth factor binding protein 2, 36 kDa NM_000597.1

TABLE 9-6 Genes that were shown to have increased expression infibroblasts as compared to the other cell lines assayed. Genes increasedin fibroblasts dual specificity phosphatase 2 KIAA0527 protein Homosapiens cDNA: FLJ23224 fis, clone ADSU02206 dynein, cytoplasmic,intermediate polypeptide 1 ankyrin 3, node of Ranvier (ankyrin G)inhibin, beta A (activin A, activin AB alpha polypeptide) ectonucleotidepyrophosphatase/phosphodiesterase 4 (putative function) KIAA1053 proteinmicrotubule-associated protein 1A zinc finger protein 41 HSPC019 proteinHomo sapiens cDNA: FLJ23564 fis, clone LNG10773 Homo sapiens mRNA; cDNADKFZp564A072 (from clone DKFZp564A072) LIM protein (similar to ratprotein kinase C-binding enigma) inhibitor of kappa light polypeptidegene enhancer in B-cells, kinase complex-associated protein hypotheticalprotein FLJ22004 Human (clone CTG-A4) mRNA sequence ESTs, Moderatelysimilar to cytokine receptor-like factor 2; cytokine receptor CRL2precursor [Homo sapiens] transforming growth factor, beta 2 hypotheticalprotein MGC29643 antigen identified by monoclonal antibody MRC OX-2putative X-linked retinopathy protein

TABLE 9-7 Genes that were shown to have increased expression in theICBM-derived cells as compared to the other cell lines assayed. GenesIncreased in ICBM Cells cardiac ankyrin repeat protein MHC class Iregion ORF integrin, alpha 10 hypothetical protein FLJ22362UDP-N-acetyl-alpha-D-galactosamine: polypeptideN-acetylgalactosaminyltransferase 3 (GalNAc-T3) interferon-inducedprotein 44 SRY (sex determining region Y)-box 9 (campomelic dysplasia,autosomal sex-reversal) keratin associated protein 1-1 hippocalcin-like1 jagged 1 (Alagille syndrome) proteoglycan 1, secretory granule

TABLE 9-8 Genes that were shown to have increased expression in the MSCcells as compared to the other cell lines assayed. Genes Increased inMSC Cells interleukin 26 maltase-glucoamylase (alpha-glucosidase)nuclear receptor subfamily 4, group A, member 2 v-fos FBJ murineosteosarcoma viral oncogene homolog hypothetical protein DC42 nuclearreceptor subfamily 4, group A, member 2 FBJ murine osteosarcoma viraloncogene homolog B WNT1 inducible signaling pathway protein 1 MCF.2 cellline derived transforming sequence potassium channel, subfamily K,member 15 cartilage paired-class homeoprotein 1 Homo sapiens cDNAFLJ12232 fis, clone MAMMA1001206 Homo sapiens cDNA FLJ34668 fis, cloneLIVER2000775 jun B proto-oncogene B-cell CLL/lymphoma 6 (zinc fingerprotein 51) zinc finger protein 36, C3H type, homolog (mouse)

TABLE 10-1 Primers used Primer name Primers Oxidized LDLS: 5′-GAGAAATCCAAAGAGCAAATGG-3′ receptor (SEQ ID NO: 1)A: 5′-AGAATGGAAAACTGGAATAGG-3′ (SEQ ID NO: 2) ReninS: 5′-TCTTCGATGCTTCGGATTCC-3′ (SEQ ID NO: 3)A: 5′-GAATTCTCGGAATCTCTGTTG-3′ (SEQ ID NO: 4) ReticulonS: 5′-TTACAAGCAGTGCAGAAAACC-3′ (SEQ ID NO: 5)A: 5′-AGTAAACATTGAAACCACAGCC-3′ (SEQ ID NO: 6) Interleukin-8S: 5′-TCTGCAGCTCTGTGTGAAGG-3′ (SEQ ID NO: 7)A: 5′-CTTCAAAAACTTCTCCACAACC-3′ (SEQ ID NO: 8) Chemokine (CXC)S: 5′-CCCACGCCACGCTCTCC-3′ ligand 3 (SEQ ID NO: 9)A: 5′-TCCTGTCAGTTGGTGCTCC-3′ (SEQ ID NO: 10)

TABLE 10-2 IL-8 protein amount measured by ELISA Cell type IL-8 hFibroND Placenta Isolate 1 ND Umb Isolate 1 2058.42 ± 144.67 Placenta Isolate2 ND Umb Isolate 2 2368.86 ± 22.73  Placenta Isolate3 (normal O₂) 17.27± 8.63 Placenta Isolate 3 (low O₂, W/O 264.92 ± 9.88  BME) Results ofthe ELISA assay for interleukin-8 (IL-8) performed on placenta- andumbilicus-derived cells as well as human skin fibroblasts. Values arepresented here are picograms/million cells, n = 2, sem. ND: Not Detected

TABLE 11-1 Antibodies Antibody Manufacturer Catalog Number HLA-DRDPDQ BDPharmingen (San Diego, CA) 555558 CD80 BD Pharmingen (San Diego, CA)557227 CD86 BD Pharmingen (San Diego, CA) 555665 B7-H2 BD Pharmingen(San Diego, CA) 552502 HLA-G Abcam (Cambridgeshire, UK) ab 7904-100 CD178 Santa Cruz (San Cruz, CA) sc-19681 PD-L2 BD Pharmingen (San Diego,CA) 557846 Mouse IgG2a Sigma (St. Louis, MO) F-6522 Mouse Sigma (St.Louis, MO) P-4685 IgG1kappa

TABLE 11-2 Mixed Lymphocyte Reaction Data - Cell Line B (Placenta) DPMfor Proliferation Assay Analytical Culture Replicates number System 1 23 Mean SD CV Plate ID: Plate1 IM03-7769 Proliferation baseline ofreceiver 79 119 138 112.0 30.12 26.9 Control of autostimulation(Mitomycin C treated autologous cells) 241 272 175 229.3 49.54 21.6 MLRallogenic donor IM03-7768 (Mitomycin C treated) 23971 22352 2092122414.7 1525.97 6.8 MLR with cell line (Mitomycin C treated cell type B)664 559 1090 771.0 281.21 36.5 SI (donor) 200 SI (cell line) 7 IM03-7770Proliferation baseline of receiver 206 134 262 200.7 64.17 32.0 Controlof autostimulation (Mitomycin C treated autologous cells) 1091 602 524739.0 307.33 41.6 MLR allogenic donor IM03-7768 (Mitomycin C treated)45005 43729 44071 44268.3 660.49 1.5 MLR with cell line (Mitomycin Ctreated cell type B) 533 2582 2376 1830.3 1128.24 61.6 SI (donor) 221 SI(cell line) 9 IM03-7771 Proliferation baseline of receiver 157 87 128124.0 35.17 28.4 Control of autostimulation (Mitomycin C treatedautologous cells) 293 138 508 313.0 185.81 59.4 MLR allogenic donorIM03-7768 (Mitomycin C treated) 24497 34348 31388 30077.7 5054.53 16.8MLR with cell line (Mitomycin C treated cell type B) 601 643 a 622.029.70 4.8 SI (donor) 243 SI (cell line) 5 IM03-7772 Proliferationbaseline of receiver 56 98 51 68.3 25.81 37.8 Control of autostimulation(Mitomycin C treated autologous cells) 133 120 213 155.3 50.36 32.4 MLRallogenic donor IM03-7768 (Mitomycin C treated) 14222 20076 2216818822.0 4118.75 21.9 MLR with cell line (Mitomycin C treated cell typeB) a a a a a a SI (donor) 275 SI (cell line) a IM03-7768 Proliferationbaseline of receiver 84 242 208 178.0 83.16 46.7 (allogenic donor)Control of autostimulation (Mitomycin treated autologous cells) 361 617304 427.3 166.71 39.0 Cell line type B Proliferation baseline ofreceiver 126 124 143 131.0 10.44 8.0 Control of autostimulation(Mitomycin treated autologous cells) 822 1075 487 794.7 294.95 37.1Plate ID: Plate 2 IM03-7773 Proliferation baseline of receiver 908 181330 473.0 384.02 81.2 Control of autostimulation (Mitomycin C treatedautologous cells) 269 405 572 415.3 151.76 36.5 MLR allogenic donorIM03-7768 (Mitomycin C treated) 29151 28691 28315 28719.0 418.70 1.5 MLRwith cell line (Mitomycin C treated cell type B) 567 732 905 734.7169.02 23.0 SI (donor) 61 SI (cell line) 2 IM03-7774 Proliferationbaseline of receiver 893 1376 185 818.0 599.03 73.2 Control ofautostimulation (Mitomycin C treated autologous cells) 261 381 568 403.3154.71 38.4 MLR allogenic donor IM03-7768 (Mitomycin C treated) 5310142839 48283 48074.3 5134.18 10.7 MLR with cell line (Mitomycin C treatedcell type B) 515 789 294 532.7 247.97 46.6 SI (donor) 59 SI (cell line)1 IM03-7775 Proliferation baseline of receiver 1272 300 544 705.3 505.6971.7 Control of autostimulation (Mitomycin C treated autologous cells)232 199 484 305.0 155.89 51.1 MLR allogenic donor IM03-7768 (Mitomycin Ctreated) 23554 10523 28965 21014.0 9479.74 45.1 MLR with cell line(Mitomycin C treated cell type B) 768 924 563 751.7 181.05 24.1 SI(donor) 30 SI (cell line) 1 IM03-7776 Proliferation baseline of receiver1530 137 1046 904.3 707.22 78.2 Control of autostimulation (Mitomycin Ctreated autologous cells) 420 218 394 344.0 109.89 31.9 MLR allogenicdonor IM03-7768 (Mitomycin C treated) 28893 32493 34746 32044.0 2952.229.2 MLR with cell line (Mitomycin C treated cell type B) a a a a a a SI(donor) 35 SI (cell line) a

TABLE 11-3 Average stimulation index of placenta cells and an allogeneicdonor in a mixed lymphocyte reaction with six individual allogeneicreceivers Average Stimulation Index Recipient Placenta Plate 1(receivers 1-3) 279 3 Plate 2 (receivers 4-6) 46.25 1.3

TABLE 11-4 Mixed Lymphocyte Reaction Data- Cell Line A (Umbilicus) DPMfor Proliferation Assay Analytical Culture Replicates number System 1 23 Mean SD CV Plate ID: Plate1 IM04-2478 Proliferation baseline ofreceiver 1074 406 391 623.7 390.07 62.5 Control of autostimulation(Mitomycin C treated autologous cells) 672 510 1402 861.3 475.19 55.2MLR allogenic donor IM04-2477 (Mitomycin C treated) 43777 48391 3823143466.3 5087.12 11.7 MLR with cell line (Mitomycin C treated cell typeA) 2914 5622 6109 4881.7 1721.36 35.3 SI (donor) 70 SI (cell line) 8IM04-2479 Proliferation baseline of receiver 530 508 527 521.7 11.93 2.3Control of autostimulation (Mitomycin C treated autologous cells) 701567 1111 793.0 283.43 35.7 MLR allogenic donor IM04-2477 (Mitomycin Ctreated) 25593 24732 22707 24344.0 1481.61 6.1 MLR with cell line(Mitomycin C treated cell type A) 5086 3932 1497 3505.0 1832.21 52.3 SI(donor) 47 SI (cell line) 7 IM04-2480 Proliferation baseline of receiver1192 854 1330 1125.3 244.90 21.8 Control of autostimulation (Mitomycin Ctreated autologous cells) 2963 993 2197 2051.0 993.08 48.4 MLR allogenicdonor IM04-2477 (Mitomycin C treated) 25416 29721 23757 26298.0 3078.2711.7 MLR with cell line (Mitomycin C treated cell type A) 2596 5076 34263699.3 1262.39 34.1 SI (donor) 23 SI (cell line) 3 IM04-2481Proliferation baseline of receiver 695 451 555 567.0 122.44 21.6 Controlof autostimulation (Mitomycin C treated autologous cells) 738 1252 464818.0 400.04 48.9 MLR allogenic donor IM04-2477 (Mitomycin C treated)13177 24885 15444 17835.3 6209.52 34.8 MLR with cell line (Mitomycin Ctreated cell type A) 4495 3671 4674 4280.0 534.95 12.5 SI (donor) 31 SI(cell line) 8 Plate ID: Plate 2 IM04-2482 Proliferation baseline ofreceiver 432 533 274 413.0 130.54 31.6 Control of autostimulation(Mitomycin C treated autologous cells) 1459 633 598 896.7 487.31 54.3MLR allogenic donor IM04-2477 (Mitomycin C treated) 24286 30823 3134628818.3 3933.82 13.7 MLR with cell line (Mitomycin C treated cell typeA) 2762 1502 6723 3662.3 2724.46 74.4 SI (donor) 70 SI (cell line) 9IM04-2477 Proliferation baseline of receiver 312 419 349 360.0 54.3415.1 (allogenic donor) Control of autostimulation (Mitomycin treatedautologous cells) 567 604 374 515.0 123.50 24.0 Cell line type AProliferation baseline of receiver 5101 3735 2973 3936.3 1078.19 27.4Control of autostimulation (Mitomycin treated autologous cells) 19244570 2153 2882.3 1466.04 50.9

TABLE 11-5 Average stimulation index of umbilicus-derived cells and anallogeneic donor in a mixed lymphocyte reaction with five individualallogeneic receivers. Average Stimulation Index Recipient UmbilicusPlate 1 (receivers 1-4) 42.75 6.5 Plate 2 (receiver 5) 70 9

TABLE 12-1 ELISA assay results MCP-1 IL-6 VEGF SDF-1α GCP-2 IL-8 TGF-β2Fibroblast 17 ± 1 61 ± 3 29 ± 2 19 ± 1 21 ± 1 ND ND Placenta 60 ± 3 41 ±2 ND ND ND ND ND (042303) Umbilicus 1150 ± 74  4234 ± 289 ND ND 160 ± 112058 ± 145 ND (022803) Placenta 125 ± 16 10 ± 1 ND ND ND ND ND (071003)Umbilicus 2794 ± 84  1356 ± 43  ND ND 2184 ± 98  2369 ± 23  ND (071003)Placenta  21 ± 10 67 ± 3 ND ND 44 ± 9 17 ± 9 ND (101503) BME Placenta 77 ± 16 339 ± 21 ND 1149 ± 137 54 ± 2 265 ± 10 ND (101503) 5% O₂, W/OBME (values presented are picograms/milliliter/million cells (n = 2,sem) Key: ND: Not Detected.

TABLE 12-2 SearchLight Multiplexed ELISA assay results TIMP1 ANG2 PDGFbbTPO KGF HGF FGF VEGF HBEGF BDNF hFB 19306.3 ND ND 230.5 5.0 ND ND 27.91.3 ND P1 24299.5 ND ND 546.6 8.8 16.4 ND ND 3.81.3 ND U1 57718.4 ND ND1240.0 5.8 559.3 148.7 ND 9.3 165.7 P3 14176.8 ND ND 568.7 5.2 10.2 NDND 1.9 33.6 U3 21850.0 ND ND 1134.5 9.0 195.6  30.8 ND 5.4 388.6 Key:hFB (human fibroblasts), P1 (placenta-derived cells (042303)), U1(umbilicus-derived cells (022803)), P3 (placenta-derived cells(071003)),U3 (umbilicus-derived cells (071003)). ND: Not Detected.

TABLE 12-3 SearchLight Multiplexed ELISA assay results MIP1a MIP1b MCP1RANTES I309 TARC Eotaxin MDC IL8 hFB ND ND 39.6 ND ND 0.1 ND ND 204.9 P179.5 ND 228.4  4.1 ND 3.8 12.2 ND 413.5 U1 ND 8.0 1694.2 ND 22.4 37.6 ND18.9 51930.1 P3 ND ND 102.7 ND ND 0.4 ND ND 63.8 U3 ND 5.2 2018.7 41.511.6 21.4 ND  4.8 10515.9 Key: hFB (human fibroblasts), P1(placenta-derived PPDC (042303)), U1 (umbilicus-derived PPDC (022803)),P3 (placenta-derived PPDC (071003)), U3 (umbilicus-derived PPDC(071003)). ND: Not Detected.

TABLE 13-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor Rat 401 (nestin) 1:200 Chemicon, Temecula, CA Human Nestin 1:100Chemicon TuJ1 (BIII Tubulin) 1:500 Sigma, St. Louis, MO GFAP 1:2000DakoCytomation, Carpinteria, CA Tyrosine 1:1000 Chemicon hydroxylase(TH) GABA 1:400 Chemicon Desmin (mouse) 1:300 Chemicon alpha -alpha-smooth 1:400 Sigma muscle actin Human nuclear 1:150 Chemiconprotein (hNuc)

TABLE 13-2 Summary of Conditions for Two-Stage Differentiation ProtocolA B COND. # PRE-DIFFERENTIATION 2^(nd) STAGE DIFF 1 NPE + F (20 ng/ml) +E (20 ng/ml) NPE + SHH (200 ng/ml) + F8 (100 ng/ml) 2 NPE + F (20ng/ml) + E (20 ng/ml) NPE + SHH (200 ng/ml) + F8 (100 ng/ml) + RA (1 μM)3 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + RA (1 μM) 4 NPE + F (20ng/ml) + E (20 ng/ml) NPE + F (20 ng/ml) + E (20 ng/ml) 5 NPE + F (20ng/ml) + E (20 ng/ml) Growth Medium 6 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 1B + MP52 (20 ng/ml) 7 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 1B + BMP7 (20 ng/ml) 8 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 1B + GDNF (20 ng/ml) 9 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 2B + MP52 (20 ng/ml) 10 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 2B + BMP7 (20 ng/ml) 11 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 2B + GDNF (20 ng/ml) 12 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 3B + MP52 (20 ng/ml) 13 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 3B + BMP7 (20 ng/ml) 14 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 3B + GDNF (20 ng/ml) 15 NPE + F (20 ng/ml) + E (20 ng/ml)NPE + MP52 (20 ng/ml) 16 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + BMP7(20 ng/ml) 17 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + GDNF (20 ng/ml)

TABLE 14-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor TuJ1 (BIII Tubulin) 1:500 Sigma, St. Louis, MO GFAP 1:2000DakoCytomation, Carpinteria, CA

TABLE 15-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor Rat 401 (nestin) 1:200 Chemicon, Temecula, CA TuJ1 (BIII Tubulin)1:500 Sigma, St. Louis, MO Tyrosine hydroxylase 1:1000 Chemicon (TH)GABA 1:400 Chemicon GFAP 1:2000 DakoCytomation, Carpinteria, CA MyelinBasic Protein 1:400 Chemicon (MBP)

TABLE 15-2 Quantification of progenitor differentiation in control vstranswell co-culture with umbilical-derived cells (E = EGF, F = bFGF)F + E/Umb F + E/F + E F + E/removed Antibody [Cond. 1] [Cond. 4] [Cond.5] TuJ1 8.7%  2.3%  3.6% GFAP 47.2% 30.2% 10.9% MBP 23.0%   0%   0%Nestin 13.4% 71.4% 39.4%

TABLE 18-1 ERG data a-wave mixed b-wave cone b-wave % rod contributionGroup Untreated Treated Untreated Treated Untreated Treated UntreatedTreated Sham 60d 0 0 7 ± 9 0 23 ± 5  12 ± 16 N/A N/A 1 (n = 4) 60d 0 20± 20 1.5 ± 2  81 ± 72 7 ± 7 50 ± 19 N/A 30 3 (n = 6) 60d 0 27 ± 11 18 ±13 117 ± 67  28 ± 11 55 ± 25 6 ± 7 49 ± 16 3 (n = 6) 90d 0 15 ± 7  0 37± 15 7 ± 5 16 ± 11 0 58 ± 39 N.B. Sham = control (medium only), 1 =Placental cell transplant, 3 = Umbilical cell transplant

TABLE 19.1 Summary of Primary Antibodies Used Antibody ConcentrationVendor Rat 401 (nestin) 1:200 Chemicon, Temecula, CA TuJ1 (BIII Tubulin)1:500 Sigma, St. Louis, MO Tyrosine hydroxylase 1:1000 Chemicon (TH)GABA 1:400 Chemicon GFAP 1:2000 DakoCytomation, Carpinteria, CA MyelinBasic Protein 1:400 Chemicon (MBP)

TABLE 19-2 Quantification of progenitor differentiation in control vstranswell co-culture with umbilical-derived cells (E = EGF, F = bFGF)F + E/Umb F + E/F + E F + E/removed Antibody [Cond. 1] [Cond. 4] [Cond.5] TuJ1 8.7%  2.3%  3.6% GFAP 47.2% 30.2% 10.9% MBP 23.0%   0%   0%Nestin 13.4% 71.4% 39.4%

TABLE 22-1 TUNEL Area Analysis at Post Natal Day 29 P29 (8 days posttreatment) Group DAPI+ area (pixels) % TUNEL+ Congenic untreated(healthy) 373,812 ± 12,832 0.2 ± 0.2% Dystrophic-untreated — —Dystrophic-sham (vehicle only) 222,016 ± 23,242 16.0 ± 2.3% Dystrophic-hUTC injected 229,666 ± 6,383  6.6 ± 0.5% (20,000 cells)

TABLE 25-1 Integrin hUTC ARPE-19 Fetal RPE * α1 − − + α2 + − + α3 + + +α4 + + + α5 + + + β1 + + + α5 β1 − − + α2β1 + + +

TABLE 26-1 Reagents used in first step in SuperScript III First-StrandSynthesis: Component: 50 μM 10 mM DEPC-treated Total RNA oligo (dT)20dNTP mix water Amount: 2 μg 2 μL 2 μL To raise volume to 20 μL

TABLE 26-2 Reagents used in cDNA Synthesis Mix: Component: 10X RT 25 mMRNaseOUT. SuperScript. buffer MgCl₂ 0.1M DTT (40 U/μl) III RT Amount: 4μL 8 μL 4 μL 2 μL 2 μL

TABLE 26-3 Reagents used in Master Mix for the RT-PCR reaction:Component: TaqMan ® Master Mix cDNA dH₂0 Gene Expression Assays Amount:10 μL 3 μL 6 μL 1 μL

TABLE 26-4 TaqMan ® gene expression assays for real time RT-PCRanalysis: Taqman Gene Abbreviation Name Expression Assay Proposedfunction in the eye CRALBP Cellular retinaldehyde-binding proteinHS00165632_ml Regeneration of visual pigment in RPE MERTK c-mer protooncogene tyrosine kinase HS00179024_ml ROS phagocytosis (ROSinternalization) GAS6 growth arrest specific-6 (ligand for MERTK)Hs00181323_ml ROS phagocytosis (ROS internalization) CD36 lipidscavenger receptor CD36 HS00169627_ml ROS phagocytosis (ROSinternalization) INTaV Integrin alpha V Hs00233808_ml ROS phagocytosis(ROS binding) INTB5 Integrin beta 5 Hs00174435_ml ROS phagocytosis (ROSbinding) CATD Cathepsin D Hs00157205_ml ROS degradation (lysosomalenzyme) CFH Complement Factor H HS00164830_ml Mediates inflammation

TABLE 26-5 Calculated □CT values for each target. Values were normalizedto GAPDH (endogenous control). Fold change is 2^(□Ct). Smaller □CTvalues represent increased gene expression. MERTK CRALBP GAS6 INTAVINTB5 CD36 CATHD CFH Untreated 16.6 ± 1.2  21 ± 3.9 3.5 ± 0.3 5.5 ± 2.22.9 ± 0.4 14.4 ± 1.0 2.1 ± 1.5 8.2 ± 0.2 hUTC 11.8 ± 0.6 14.3 ± 2.6 5.0± 1.1 2.5 ± 0.5 3.0 ± 1.5 15.3 ± 1.3 0.3 ± 0.6 2.8 ± 0.2 Treament

TABLE 27-4 Trophic Factor Secretion by hUTC Sample hUTC hUTC hFIb. hFib.FGF-b pg/ml/million 64.06 74.00 7.43 7.64 HGF pg/ml/million 980.23703.24 37.76 30.54 KGF pg/ml/million 50.78 41.15 81.28 63.43 VEGFpg/ml/million 118.00 90.80 202.52 165.96 GRO-a pg/ml/million 3758.522398.48 795.43 760.72 MCP1 pg/ml/million 948.56 625.71 218.83 182.95GMCSF pg/ml/million 400.16 267.54 129.11 150.81 IL6 pg/ml/million 103.5769.23 75.39 55.46 IL8 pg/ml/million 7308.60 3832.09 3843.18 2781.39 TNFapg/ml/million 21.72 6.91 6.91 6.91 B-NGF pg/ml/million 5.16 1.18 10.068.28 BDNF pg/ml/million 525.38 432.08 220.41 191.69 CNTF pg/ml/million102.11 74.58 55.06 10.00 NT-3 pg/ml/million 33.29 16.78 22.87 21.38NT-proBN pg/ml/million 24.14 21.91 14.92 12.56 TGFb pg/ml/million 662.45718.24 1144.50 298.62

TABLE 27-5 IGF Secretion buy hUTC IGF(ng/ml/million) SD hUTC 43529.4143.53 Fib 24759.26 24.70

1.-27. (canceled)
 28. A method for promoting axonal regeneration in anoptic nerve in a patient having an ocular degenerative conditioncomprising administering to the optic nerve isolated umbilicus-derivedcells isolated from human umbilical cord tissue substantially free ofblood, in an amount effective to promote axonal regeneration, whereinthe isolated umbilicus cells are capable of self-renewal and expansionin culture, have the potential to differentiate into cells of at least aneural phenotype, maintain a normal karyotype upon passaging and lackproduction of CD117.
 29. The method of claim 28, wherein the isolatedumbilicus-derived cells further have the following characteristics: a)potential for at least 40 population doublings in culture; b) productionof CD10, CD13, CD44, CD73, and CD90; c) lack of production of CD31,CD34, CD45, and CD141; and d) increased expression of genes encodinginterleukin 8 and reticulon 1 relative to a human cell that is afibroblast, a mesenchymal stem cell, or an iliac crest bone marrow cell.30. The method of claim 29, wherein the isolated umbilicus cells arepositive for HLA-A, B, C, and negative for HLA-DR, DP, DQ.
 31. Themethod of claim 28, wherein the umbilicus-derived cells are expanded inculture prior to administering to the patient's eye.
 32. The method ofclaim 28, wherein the umbilicus-derived cells are administered with atleast one other agent.
 33. The method of claim 32, wherein the at leastone other agent is administered simultaneously with, or before, orafter, the umbilicus-derived cells.
 34. The method of claim 28, whereinthe umbilicus-derived cells are administered through a cannula or from adevice inserted in the patient's eye.
 35. The method of claim 28,wherein the umbilicus-derived cells are administered by insertion of amatrix or scaffold containing the cells.
 36. A method for increasingthickness of the outer nuclear layer of a retina in a patient having anocular degenerative condition comprising administering isolatedumbilicus-derived cells isolated from human umbilical cord tissuesubstantially free of blood, in an amount effective to promote axonalregeneration, wherein the isolated umbilicus cells are capable ofself-renewal and expansion in culture, have the potential todifferentiate into cells of at least a neural phenotype, maintain anormal karyotype upon passaging and lack production of CD117.
 37. Themethod of claim 36, wherein the isolated umbilicus-derived cells furtherhave the following characteristics: a) potential for at least 40population doublings in culture; b) production of CD10, CD13, CD44,CD73, and CD90; c) lack of production of CD31, CD34, CD45, and CD141;and d) increased expression of genes encoding interleukin 8 andreticulon 1 relative to a human cell that is a fibroblast, a mesenchymalstem cell, or an iliac crest bone marrow cell.
 38. The method of claim37, wherein the isolated umbilicus cells are positive for HLA-A, B, C,and negative for HLA-DR, DP, DQ.
 39. The method of claim 36, wherein theumbilicus-derived cells are expanded in culture prior to administeringto the patient's eye.
 40. The method of claim 36, wherein theumbilicus-derived cells are administered with at least one other agent.41. The method of claim 40, wherein the at least one other agent isadministered simultaneously with, or before, or after, theumbilicus-derived cells.
 42. The method of claim 36, wherein theumbilicus-derived cells are administered through a cannula or from adevice inserted in the patient's eye.
 43. The method of claim 36,wherein the umbilicus-derived cells are administered by insertion of amatrix or scaffold containing the cells.
 44. A kit for treating apatient having an ocular degenerative condition comprising apharmaceutical acceptable carrier, isolated umbilicus-derived cellsisolated from human umbilical cord tissue substantially free of bloodand instructions for use, wherein the isolated umbilicus cells arecapable of self-renewal and expansion in culture, have the potential todifferentiate into cells of at least a neural phenotype, maintain anormal karyotype upon passaging and lack production of CD117.
 45. Thekit of claim 44, wherein the isolated umbilicus-derived cells furtherhave the following characteristics: a) potential for at least 40population doublings in culture; b) production of CD10, CD13, CD44,CD73, and CD90; c) lack of production of CD31, CD34, CD45, and CD141;and d) increased expression of genes encoding interleukin 8 andreticulon 1 relative to a human cell that is a fibroblast, a mesenchymalstem cell, or an iliac crest bone marrow cell.
 46. The kit of claim 44,wherein the isolated umbilicus cells are positive for HLA-A, B, C, andnegative for HLA-DR, DP, DQ.
 47. The kit of claim 44, further comprisinga cannula for administering the cells.